Dna Guide

www.promega.com techserv@promega.
com
Table of Contents
Genomic DNA Purification 2
Overview
Wizard
Genomic DNA Purification Kit

Wizard
SV and SV 96 Genomic DNA Purification Systems

MagneSil Blood Genomic, Max Yield System
MagneSil ONE, Fixed Yield Blood Genomic System
Wizard
Magnetic 96 DNA Plant System

Ordering Information
Amplifying DNA 12
Overview
Optimization of PCR
Routine PCR
Proofreading Polymerases
Hot Start Methodology
dNTPs
PCR Clean-Up 26
Overview
Wizard
SV Gel and PCR Clean-Up System

Wizard
SV 96 PCR Clean-Up System

Wizard
MagneSil PCR Clean-Up System

Wizard
PCR Preps DNA Purification System

Cloning PCRDNA 32
Overview
Basic Subcloning
Direct Mammalian Expression
Direct Bacterial Expression
PCR Cloning Techniques
DNA Analysis
Notebook
Promega DNA Analysis Notebook
2
Genomic DNA Purification
Overview
Promega has a variety of solutions for your genomic
DNA (gDNA) purification needs. We provide manual and
automated solutions for DNA purification from blood,
cultured cells, mammalian tissue and plant tissue. All kits
produce high-quality DNA that is ready for amplification.
Our MagneSil Paramagnetic Particle
(a)
technology
provides automated solutions for isolation of gDNA from
blood or plant tissue. The Wizard
SV technology
introduces membrane-based purification for manual or
automated procedures, and the Wizard
Genomic DNA
Purification Kit provides a versatile solution-based
system for the manual isolation of gDNA from all the
above materials and from bacterial and yeast cells. This
gentle, solution-based method produces DNA suitable for
amplification, Southern blotting and genomic cloning.
Look for these symbols to find the right system
for your application.
Blood
Cultured Cells
Animal Tissue
Plant Tissue
Bacteria
Yeast
0.050.2ml 0.210ml
Manual
MagneSil ONE,
Fixed Yield Blood
Genomic System
3
9
8
1
M
A
0
2
_
3
A
Automated
Wizard
Genomic
DNA Purification Kit
MagneSil Blood
Genomic, Max
Yield System
Blood
Please Note:
In the flowcharts that follow, DNA purification
systems are listed by application (blood, cultured
cells, tissue, etc.) based on the protocols given in
the Technical Manuals provided with each system.
Applications for each system with other gDNA
sources may exist (e.g., protocols for DNA
purification from blood using the Wizard
SV
Genomic DNA Purification System). Please contact
Promega Technical Services if you have questions.
techserv@promega.com
B
u
f
f
y

c
o
a
t

o
r

w
h
it
e

b
lo
o
d

c
e
lls
?

U
s
e

c
u
lt
u
r
e
d

c
e
lls

p
r
o
t
o
c
o
ls
.
S
olution-based, totally
scalable system
. U
ses
centrifugation. S
pecific
protocols provided for
30
0
l, 3m
l and 10
m
l of
fresh w
hole blood.
Requires automation.
Magnetic-based system.
Handles 200l of blood.
Maximizes yield
from sample.
R
equires autom
ation. M
agnetic
-
based system
. D
esigned to capture ~
1g
gD
N
A
from
5
0
60
l of blood.
E
lim
inates need to quantitate after purification.
www.promega.com techserv@promega.com
3
Manual
Wizard
SV 96
Genomic DNA
Purification System
3
9
8
2
M
A
0
2
_
3
A
Automated
Wizard
Genomic
Wizard
SV
Genomic DNA
Purification System
3
9
8
5
M
A
0
2
_
3
A
Manual
Wizard
Genomic
Manual
You supply
lyticase and
50mM EDTA
You supply
lysozyme and/or
lysostaphin
Gram-
negative
Gram-
positive
Cultured
Cells
Yeast Bacteria
S
o
l
u
t
i
o
n
-
b
a
s
e
d
,

t
o
t
a
l
l
y
s
c
a
la
b
le

s
y
s
t
e
m
.
U
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e
s
c
e
n
t
r
if
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g
a
t
i
o
n
.
P
r
o
d
u
c
e
s
h
i
g
h

m
o
le
c
u
la
r

w
e
i
g
h
t
(
>
5
0
k
b
)

g
D
N
A

s
u
i
t
a
b
le
f
o
r

a
n
y

a
p
p
l
i
c
a
t
i
o
n
,
i
n
c
l
u
d
i
n
g
P
C
R

a
n
d
S
o
u
t
h
e
r
n

b
lo
t
t
i
n
g
.
U
se
w
ith
a
m
ic
roc
e
ntrifug
e
or vac
uum
m
anifold
and
vac
uum
ad
apte
rs. G
e
t P
C
R
-
re
ad
y
g
D
N
A
in 2
0
m
inute
s
afte
r lysis. C
an proc
e
ss up
to 5
x 10
6
c
e
lls/pre
p.
Perform 96 gDNA preps
at once. Requires a vacuum
manifold. Can be used on
benchtop or automated.
Comprised of SV
membranes arrayed in a
96-well format. Can
handle up to 5 x 10
6
cells/well.
S
olution-
base
d
,
totally
sc
alable

sy
ste
m
w
ith

protoc
ols
for
bac
te
ria
and

y
e
ast.
U
se
s
c
e
nt
rifug
ation.
P
rod
uc
e
s
h
ig
h
m
ole
c
ular
w
e
ig
h
t
g
D
N
A

suitable

for
any
applic
ation,
inc
lud
ing
P
C
R

and
S
outh
e
rn
blotting
.
4
Genome Size Molecules/g
Human 2.9Gb 3.13 10
5
Mouse 2.7Gb 3.35 10
5
Rat 2.8Gb 3.26 10
5
Arabidopsis 3.0Gb 3.04 10
5
Tobacco 4.4Gb 2.07 10
5
Corn 2.5Gb 3.65 10
5
Wheat 16.0Gb 5.70 10
4
Yeast (S. cerevisiae) 13.5Mb 6.75 10
7
Bacteria (E. coli ) 4.7Mb 1.94 10
8
Gb = Gigabases (1 10
9
). Mb = Megabases (1 10
6
).
Manual
Wizard
Magnetic
96 DNA
Plant System
3
9
8
4
M
A
0
2
_
3
A
Automated
Wizard
Genomic
Requires a 96-well
grinding apparatus
Requires liquid
nitrogen, mortar & pestle
Plant
Tissue
S
olution-
base
d
, totally
sc
alable
syste
m
for plant
tissue. U
se
s
c
e
ntrifug
ation. P
rod
uc
e
s
h
ig
h
m
ole
c
ular w
e
ig
h
t
(>
5
0
k
b) g
D
N
A
suitable
for any applic
ation,
inc
lud
ing
P
C
R
and
S
outh
e
rn blotting
.
M
agnetic-based system
that can be used
in m
anual or autom
ated form
at. Purifies
gD
N
A
from
seeds or tissues. Processes
8
m
m
leaf punches, 1
5
seeds. R
equires a
M
agnaB
ot
9
6 M
agnetic S
eparation D
evice (purchase separately).
Dont see your DNA sample
type in these flowcharts?
Contact Promega Technical
Services for advice at:
techserv@promega.com
5
Manual
Wizard
SV 96
Genomic DNA
Purification System
3
9
8
3
M
A
0
2
_
3
A
Automated
Wizard
Genomic
Wizard
SV
Genomic DNA
Purification System
Overnight
proteinase K
digestion
(you supply the enzyme)
Overnight
proteinase K
digestion
(you supply the enzyme)
Animal
Tissue
P
e
rf
o
r
m
s
9
6

g
D
N
A
pr
e
p
s
a
t

o
n
c
e
.
U
se
s
a
v
a
c
uum

m
a
n
if
old
.
C
a
n
b
e

use
d

o
n

t
h
e
b
e
n
c
h
top
o
r

a
uto
m
a
te
d
.
C
o
m
pr
ise
d

o
f

S
V
m
e
m
b
r
a
n
e
s
a
r
r
a
y
e
d

in
a

9
6
-
w
e
ll
f
o
r
m
a
t.
P
r
o
c
e
sse
s
up
to

2
0
m
g t
issue
/
w
e
ll.
Use with a
microcentrifuge or vacuum
manifold with vacuum
adapters. Get PCR-ready
gDNA in 20 minutes
after lysis. Can process up
to 20mg tissue/prep.
S
o
lu
t
io
n
-
b
a
s
e
d
,
t
o
t
a
lly

s
c
a
la
b
le
s
y
s
t
e
m
.
U
s
e
s

c
e
n
t
r
if
u
g
a
t
io
n
.
P
r
o
d
u
c
e
s

h
ig
h

m
o
le
c
u
la
r

w
e
ig
h
t
(
>
5
0
k
b
)

g
D
N
A

s
u
it
a
b
le

f
o
r

a
n
y
a
p
p
lic
a
t
io
n
,
in
c
lu
d
in
g
P
C
R

a
n
d
S
o
u
t
h
e
r
n

b
lo
t
t
in
g
.
6
Wizard
Genomic DNA
Purification Kit
Need a versatile genomic DNA purification system?
Get it all with the Wizard

Kit. This solution-based system uses a simple, gentle
salting out method to isolate genomic DNA from a
wide variety of starting materials. The standard protocol
allows isolation of gDNA from blood or cultured cells.
Simple modifications to the protocol (involving the
addition of reagents common to most molecular
biology laboratories) allow you to isolate gDNA from
bacteria, yeast, plant or animal tissues, including
mouse tails. The system isolates high molecular weight
DNA (>50kb) with an A
260
/A
280
greater than 1.7.
Protocols provided for:
Whole Blood
(300l, 3ml, 10ml, and 50l 96 wells)
Cultured Cells
Animal Tissue
Plant Tissue
Gram-Negative Bacteria
Gram-Positive Bacteria
Yeast
DNA Yields from Various Starting Materials.
Amount of Typical
Source Starting Material DNA Yield
Whole Blood 300l 515g
3ml 2550g
10ml 250500g
96-well plate, 50l/well 0.20.7g
Tissue Culture Cells 10
6
10
7
cells 530g
Animal Tissue
Mouse Liver 11mg 1520g
Mouse Tail 0.51cm of tail 1030g
Insect Cells 5 10
6
cells 16g
Plant Leaf Tissue 40mg 712g
Bacterial Culture* 10
8
10
10
cells 520g
Yeast* 1.9 10
8
cells 4.56.5g
*Overnight culture.
Genomic DNA isolated using the Wizard
Genomic DNA Purification Kit.

Genomic DNA was isolated from fresh rat brain or PC12 cells according to the protocol
provided in the Wizard
Genomic DNA Purification Kit Technical Manual #TM050. DNA

from the indicated sources (0.5g/lane) was separated on a 0.7% agarose gel.
Wizard

Cat.#: A1120 (100 isolations, 300l blood)
A1125 (500 isolations, 300l blood)
A1620 (100 isolations, 10ml blood)
Protocol:
www.promega.com/tbs/tm050/tm050.html
Customizable Protocol:
www.promega.com/tbscustom/tm050c/promega.asp
Citations detailing use of this kit:
www.promega.com/citations/
The Wizard
Genomic DNA Purification Kit has been

cited for purification of gDNA from the following
bacterial sources:
Bordetella, Borrelia, Campylobacter, Desulfovibrio,
Escherichia, Flavobacterium, Haemophilus,
Helicobacter, Leptospira, Methanococcus,
Mycobacterium, Mycoplasma, Paracoccus,
Salmonella, Serratia, Sphingomonas, Staphylococcus,
Streptococcus and Vibrio.
Citations describing isolation from these and other
sources, including yeast, fungi and virus-infected
cells, are available online at:
1
0
1
0
M
A
0
4
_
5
B
L
a
m
b
d
a

D
N
A
,

u
n
c
u
t
L
a
m
b
d
a

D
N
A
P
C
1
2

c
e
ll
D
N
A
,

u
n
c
u
t
R
a
t

b
r
a
in

D
N
A
,

u
n
c
u
t
T
h
e

W
iz
a
r
d
G
e
n
o
m
ic
D
N
A
P
u
r
if
ic
a
t
io
n

K
it
p
r
o
v
id
e
s

p
r
o
t
o
c
o
ls

f
o
r

t
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e
e
a
s
y
,
s
o
lu
t
io
n
-
b
a
s
e
d
,
m
a
n
u
a
l
p
u
r
if
ic
a
t
io
n

o
f

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D
N
A

f
r
o
m
m
a
n
y

d
if
f
e
r
e
n
t

s
o
u
r
c
e
s
.
7
Wizard
SV and SV 96
Systems
The Wizard
SV and SV 96 Genomic DNA Purification

Systems provide a fast, simple technique for the
preparation of genomic DNA from cultured cells and
tissue, including mouse tails. The SV system is
designed for single-prep manual applications using
either a microcentrifuge or vacuum manifold. Genomic
DNA is obtained in 20 minutes after cell or tissue lysis.
The SV 96 system was developed to meet high-
throughput needs. You can use this system on the
benchtop for manual 96-well purifications or automate
on a liquid-handling platform like the Beckman Coulter
Biomek
FX or Biomek
2000 with a suitable vacuum

manifold. Both systems provide similar yields of high
quality, PCR-ready genomic DNA. Isolation of DNA from
tissue requires the additional purchase of DNase-free
Proteinase K (e.g., Promega Cat.# V3021).
M C +C S
p
i
n

T
a
i
l
S
p
i
n
L
i
v
e
r
S
p
i
n
H
e
a
r
t
S
p
i
n
B
r
a
i
n
V
a
c
u
u
m

T
a
i
l
V
a
c
u
u
m

L
i
v
e
r
V
a
c
u
u
m

H
e
a
r
t
V
a
c
u
u
m

B
r
a
i
n
3
7
1
0
T
A
0
4
_
2
A
Amplification of genomic DNA isolated from various mouse tissue sources
using the Wizard
SV Genomic DNA Purification System. Genomic DNA was

isolated from the tissues listed using either the vacuum or spin protocols provided in the
Wizard
SV Genomic DNA Purification System Technical Bulletin #TB302. One microliter of

the eluate from the column was amplified for a mouse IL-1 (1.2kb) product. The positive
control (+C) was Mouse Genomic DNA (Cat.# G3091) and the negative control (C)
contained no DNA. Further details are provided in Grunst, T. and Worzella, T. (2002)
Introducing the Wizard
SV and SV 96 Genomic DNA Purification Systems. Promega Notes

81, 913.
Want to use the SV Genomic Systems for manual
gDNA purification from blood?
Request Genomic DNA Purification from Blood:
Wizard
SV Genomic DNA Purification Systems.

Application Note #AN101:
Overview of the Wizard
SV Genomic DNA Purification System spin and

vacuum protocols. *Vacuum Adaptor, Vacuum Manifold and Proteinase K must be
purchased separately.
3
6
4
3
M
A
0
2
_
2
A
Add Wizard
SV Lysis Buffer.
Centrifuge.
Bind DNA.
Wash, removing
solution by
centrifugation
or vacuum.
Transfer spin
column to a 1.5ml
microcentrifuge
tube (not provided).
Centrifuge.
Mouse tail clipping
or tissue sample
Proteinase K
(Cat.# V3021)*
digestion in
Digestion Solution.
Genomic DNA
Purification from
Mouse Tail Clipping
or Tissue Sample
Genomic DNA
Purification from
Tissue Culture Cells
Wash tissue
culture cells
with 1X PBS.
Incubate at 55C
overnight
(1618 hours).
Add Wizard
SV Lysis Buffer.
Transfer lystate to minicolumn.
Elute genomic DNA.
Vacuum Adapter
(Cat.# A1331)*
Vac-Man
Laboratory
Vacuum Manifold
(Cat.# A7231)*
Wizard
SV Genomic DNA Purification System

Cat.#: A2360 (50 preps)
A2361 (250 preps)
Protocol:
www.promega.com/tbs/tb302/tb302.html
8
Average Yield of Genomic DNA Purified from Various
Sources Using the Wizard
SV and SV 96 Genomic DNA

Purification Systems.
Sample Type Starting Amount Average Yield
Mouse Tail Clipping 20mg 20g
Mouse Liver 20mg 15g
Mouse Heart 20mg 10g
Mouse Brain 20mg 6g
CHO Cells 1 10
6
cells 5g
NIH3T3 Cells 1 10
6
cells 9g
293 Cells 1 10
6
cells 8g
Comparison of DNA yields using the Wizard
SV and SV 96 Genomic DNA

Purification Systems. Average yield of genomic DNA (g) purified from 20mg mouse
tail clippings (1.2cm tail tip portions). Average A
260
/A
280
ratios are: SV 96, 1.7 0.08;
SV Vacuum, 1.7 0.14; and SV Spin, 1.7 0.14.
Cross-contamination assay. Genomic DNA was purified from mouse tail clippings or
water samples arrayed in adjacent wells of a 96-well plate using the Wizard
SV 96
Genomic DNA Purification System. PCR products were amplified from 1l of purified
sample from each well for mouse IL-1 (1.2kb). No product is expected from wells
containing water. For further details, see Grunst, T. and Worzella, T. (2002) Introducing the
Wizard
SV and SV 96 Genomic DNA Purification Systems. Promega Notes 81, 913.

0
5
10
15
20
25
30
35
0
5
10
15
20
25
30
35
A
v
e
r
a
g
e

Y
i
e
l
d

(
g
)
Method of Purification
SV 96 SV Vacuum SV Spin
M C +C C4 C5 C6 C7 C8 D4 D5 D6 D7 D8 M E4 E5 E6 E7 E8
3
7
1
2
T
A
0
4
_
2
A
Mouse Tail Clippings
Water Only
1 2 3 4 5 6 7 8 9 10 11 12
A
B
C
D
E
F
G
H
Wizard
SV 96 Genomic DNA
Purification System
Cat.#: A2370 (1 96 preps)
A2371 (4 96 preps)
Protocol:
The Wizard
SV 96 Genomic System is suitable for

manual DNA purification at the benchtop or can be
easily automated on liquid handlers such as the
Biomek
FX, Biomek
2000, or MultiPROBE
II HT/EX.
For more information visit:
www.promega.com/automethods/
3
4
4
6
C
A
0
6
_
1
A
W
ork
w
ith the term
inal 2cm
of
m
ouse tails. A
ny higher up on the tail
and you'll get m
ore connective tissue,
cartilage and bone than nucleated cells.
T
his m
aterial not only clogs colum
ns,
but few
er cells m
eans less gD
N
A
.
R
o
b
o
t

t
u
r
n
s

i
n
t
o

a

g
e
n
o
m
i
c

D
N
A

m
a
c
h
i
n
e
!
9
MagneSil Blood Genomic,
Max Yield & MagneSil ONE,
Fixed Yield Blood Genomic
Promega has developed two new genomic DNA
isolation systems to streamline your blood-to-analysis
pathwayMagneSil Blood Genomic, Max Yield
(a)
and
MagneSil ONE, Fixed Yield Blood Genomic
(a)
. Both
systems are designed for automated gDNA purification
on liquid-handling workstations such as the Biomek
FX.
The Max Yield System purifies 46g of gDNA from
200l of whole blood. The MagneSil ONE System
purifies 1g gDNA (50%) from 5060l whole blood.
Both systems produce gDNA that is ready for use in
PCR or multiplex amplification reactions.
Cat.#: MD1370 (1 96 preps)
Protocol:
For more information on automated methods visit:
Cat.#: MD1360 (1 96 preps)
Protocol:
Purify up to 36g
gDNA from 200l
whole blood.
DNA Yields (ng) From 60l Whole Blood Samples Using
the MagneSil ONE, Fixed Yield Blood Genomic System.
Donor 1 Donor 2 Donor 3 Donor 4 Donor 5
Sample 1 1078 1078 1231 1451 1025
Sample 2 990 1092 1256 1078 998
Sample 3 970 1047 1315 990 994
Sample 4 1209 1294 1047 970 967
Sample 5 1105 1063 1388 1209 843
Sample 6 839 843 1296 1105 797
Mean 1032 1070 1256 1134 937
SD 128 143 116 178 94
4
0
3
1
C
A
0
3
_
3
A
Analysis of DNA isolated using the MagneSil Blood Genomic, Max Yield System. DNA isolated from whole blood using the MagneSil Blood Genomic, Max Yield System was
used with the PowerPlex
16 System (Cat.# DC6531), a multiplex STR amplification system for use in DNA typing. Results show successful coamplification and 3-color detection of the 16 loci
(15 STR loci plus amelogenin) in the PowerPlex
16 System
(b,c,d)
. Amplification products were separated on an ABI PRISM
310 Genetic Analyzer, and analyzed using GeneScan
Software.
Purify ~1g gDNA
from 5060l
whole blood.
F
ixe
d

Y
ie
ld

m
e
an
s
n
o
m
ore
quan
titation

or

n
or
m
aliz
ation
!
O
ptim
iz
e

t
h
e

v
olum
e

of
e
lute
d

D
N
A

y
ou
n
e
e
d on
c
e
use

t
h
at

sam
e
am
oun
t

e
ac
h

tim
e
.
Wizard
Magnetic 96 DNA
Plant System
The Wizard

(a)
is
designed for manual or automated 96-well purification
of genomic DNA from plant leaf and seed tissue. The
system was validated with corn and tomato leaf as well
as with canola and sunflower seeds. The DNA purified
from these samples can be used in PCR as well as more
demanding applications such as Rapid Amplification of
Polymorphic DNA (RAPD) analysis. The Wizard
Magnetic 96 DNA Plant System uses MagneSil

Paramagnetic Particles
(a)
(PMPs), considered a mobile
solid phase. The binding of nucleic acids to magnetic
particles occurs in solution, resulting in increased
binding kinetics and binding efficiency. Contact with the
wash buffer is also enhanced, facilitating removal of
contaminants and increasing nucleic acid purity.
Automated methods for the Beckman Biomek
2000,
Biomek
FX and other robots are available.

10
The Wizard
Magnetic 96 DNA Plant System produces PCR-quality DNA from

a variety of plant species. One microliter of gDNA purified from the indicated materials
was used as template in PCR using a universal primer pair specific for the intron of the
TrnL chloroplast gene. One or more bands are produced depending on the plant species.
For more information, please see Koller, S. et al. (2001) Automated genomic DNA
purification using the Wizard
Magnetic 96 DNA Plant System. Promega Notes 79, 2528.

Plant Sample Types Processed Using the Wizard
Magnetic 96 DNA Plant System.

Arapidopsis Cotton seed Soybean
Cabbage seed Grass seed Squash
Canola leaf Green pepper seed Squash seed
Canola seed Lettuce Strawberry leaf*
Carrot seed Milkweed leaf Sunflower seed
Chicory leaf Potato tuber Tobacco seedling
Chives Radish leaf Tomato leaf
Corn leaf Rice leaf Tomato seed
Cotton leaf* Sorghum Watermelon seed
*These samples require addition of polyvinylpolypyrrolidone (PVPP) to the
lysis buffer to remove phenolic compounds that inhibit PCR.
Typical DNA Yield from Plant Species Using the Wizard
Magnetic 96 DNA Plant System.

Arabidopsis tissue 10ng/mg
Canola leaf punches* (12) 26ng/leaf punch
Canola seeds (5) 343ng/seed
Corn leaf punches* (12) 98ng/leaf punch
Cotton seed (1) 29ng/seed
Lettuce leaf punches* (8) 13ng/leaf punch
Melon seed (1) 166ng/seed
Radish leaf punches* (12) 89ng/leaf punch
Soybean (10mg) 10ng/mg bean
Squash seed (1) 279ng/seed
Sunflower seed (1) 405ng/seed
Tomato leaf punches* (12) 111ng/leaf punch
*Leaf punches 6mm in diameter.
100
500
1,500
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
1
2
3
4
5
6
7
8
Tobacco seedling
Soybean
Lettuce leaf
Corn leaf
Potato
Arabidopsis leaf
Cabbage seed
Green pepper seed
9
10
11
12
13
14
15
Prairie grass, seed head
Chives
Tomato leaf
Rice
Canola seed
Sunflower seed
Carrot seed
bp
3
3
1
5
T
B
0
3
_
1
A
Wizard

Cat.#: FF3760 (2 96 preps)
FF3761 (4 96 preps)
Protocol:
Want to use the Wizard
Magnetic 96 DNA Plant

System for high yield or fixed yield gDNA
purification from plant tissue?
Request Application Notes #AN104 and #AN105
11
Genomic DNA Purification Systems and Accessories
Product Size Cat.#
Wizard
Genomic DNA Purification Kit 100 isolations (300l blood per isolation) A1120
500 isolations (300l blood per isolation) A1125
100 isolations (10ml blood per isolation) A1620
For Laboratory Use. Cat.# A1120 will give ~40 animal tissue preps, ~80 mouse tail preps, ~80 plant tissue preps, and ~80 cultured cell preps. Please see the Wizard

Technical Manual #TM050 for more details and additional supplies necessary for the various preps.
Product Size Cat.#
Wizard
SV Genomic DNA Purification System 50 preps (20mg tissue per prep) A2360
250 preps (20mg tissue per prep) A2361
Vac-Man
Laboratory Vacuum Manifold, 20-sample capacity 1 each A7231

Vacuum Adapters 20 each A1331
The Wizard
SV Genomic DNA Purification System can be used in spin or vacuum format. The Vac-Man
Laboratory Vacuum Manifold can be used to process up to 20 samples at once. The

Vacuum Adapters are required when using the the Vac-Man
Laboratory Vacuum Manifold with the Wizard
SV Genomic DNA Purification System.

Product Size Cat.#
Wizard
SV 96 Genomic DNA Purification System 1 96 preps (20mg tissue per prep) A2370
4 96 preps (20mg tissue per prep) A2371
Vac-Man
96 Vacuum Manifold 1 each A2291

The Vac-Man
96 Vacuum Manifold is required for use with the Wizard
SV 96 Genomic DNA Purification System.

Product Size Cat.#
(a)
* 1 96 preps (5060l blood per prep) MD1370
(a)
* 1 96 preps (200l blood per prep) MD1360
Deep Well MagnaBot
96 Magnetic Separation Device* 1 each V3031

MagnaBot
Spacer, 1/8 inch 1 each V8581

* For Laboratory Use. Both MagneSil Blood Genomic Systems require use of an automated liquid handler such as the Beckman Biomek
FX. The Deep Well MagnaBot
96 Magnetic Separation
Device and the MagnaBot
Spacer, 1/8 inch, are also required for use with these systems.
Product Size Cat.#
Wizard

(a)
2 96 preps FF3760
4 96 preps FF3761
MagnaBot
96 Magnetic Separation Device 1 each V8151

MagnaBot
Spacer 1 each V8381

The Wizard
Magnetic DNA Plant System requires use of the MagnaBot
96 Magnetic Separation Device and the MagnaBot
Spacer for manual or automated DNA purification.

Related Products
Product Size Cat.#
Proteinase K 100mg V3021
RNase A Solution, 4mg/ml 1ml A7973
For Laboratory Use. Proteinase K is required for tissue preparations. RNase A is certified DNase-free and is used to remove RNA from gDNA preps.
12
Amplifying DNA
Overview
Denature Anneal Extend PCR amplification led to a
revolution in molecular biology in the 1980s. PCR is a
relatively simple technique by which a DNA or cDNA
template is amplified many thousand- or millionfold
quickly and reliably, generating sufficient material for
subsequent analyses.
The PCR process is exquisitely sensitive. While most
biochemical analysesincluding nucleic acid detection
with radioisotopesrequire the input of significant
amounts of biological material, the PCR process
requires very little starting material. This feature makes
the technique extremely useful, not only in basic
research, but also for applications such as genetic
identity testing, forensic analysis, industrial quality
control and in vitro diagnostics.
The availability of such a powerful tool has led to
significant developments in answering biological
questions. Many adaptations of the original PCR method
have been published, and numerous factors that are
critical for accurate amplification have been identified.
How Much Enzyme is Needed in a Reaction?
Promega recommends using 1.25 units of
thermostable DNA polymerase per 50l amplification
reaction. For most applications, the enzyme will be in
excess. The inclusion of more enzyme will not
significantly increase yield. Increased amounts of
enzyme and excessively long extension times will
increase the likelihood of artifacts due to the intrinsic
53 exonuclease activity of Taq DNA Polymerase
(e)
and other non-proofreading DNA polymerases.
Artifacts are generally seen as smeared bands in
ethidium bromide-stained agarose gels.
The most frequent cause of excessive enzyme levels
is pipetting error. Accurate dispensing of
submicroliter (<1l) volumes of enzyme solutions in
50% glycerol is nearly impossible. We strongly
recommend making reaction master mixes sufficient
for the number of reactions being performed. A
master mix increases the volume of pipetted reagents
and reduces pipetting errors.
Typical Reaction with Taq DNA Polymerase.
Nuclease-Free Water to 50l final
Reaction Buffer(10X or 5X) 1X
dNTPs 0.2mM each
Taq DNA Polymerase 1.25u
MgCl
2
* 0.54.0mM
Downstream primer 1M (50pmol)
Upstream primer 1M (50pmol)
Template 10
4
copies
*Some reaction buffers contain Mg
2+
, and additional MgCl
2
may not be required.
The optimal Mg
2+
concentration depends on the template but is typically in the range
0.54mM.
Assemble reactions on ice in the order listed. Be sure to vortex the MgCl
2
solution,
primers, dNTPs and Reaction Buffer prior to addition. When using a thermal cycler without
a heated lid, overlay the reaction with 12 drops of mineral oil to prevent evaporation.
Setting up reactions with a
proofreading polymerase?
Proofreading enzymes like to chew on
free primers due to their 3'5'
exonuclease activity. Always assemble
the reaction on ice and add the
proofreading polymerase last,
just prior to placing the tubes in a
preheated 9495C thermal cycler.
See p. 21 for more information.
For more
information on
reaction
optimization,
see pp. 1417.
V
or
te
x
all
M
g
C
l
2
-
c
ontaining
solutions
th
oroug
h
ly
prior
to
use
!
M
g
C
l
2
solutions
c
an
form

a
c
onc
e
nt
ration
g
rad
ie
nt
upon
th
aw
ing
.
F
ailure

to
vor
te
x
is
a
c
om
m
on
sourc
e

of
P
C
R

failure
!
13
Amplifying DNA
Example Cycling Conditions for Taq DNA Polymerase.
Time
Step Temperature (minutes) Cycles
Initial
Denaturation
(a)
95C 2 1
Denaturation 95C 0.51
Annealing 4265C
(b)
0.51 2535
Extension 72C 1 min/kb
(b,c)
Final Extension 72C 5 1
Soak 4C indefinite 1
(a)
Reactions are placed in a thermal cycler that has been preheated to 95C. The thermal
cycling protocol has an initial denaturation step where samples are heated at 95C for
2 minutes to ensure that the target DNA is completely denatured. Initial denaturation of
longer than 2 minutes at 95C is usually unnecessary and may reduce yield. (Some hot
start polymerases require pre-incubation at 95C to activate the polymerase prior to the
2-minute denaturation step.)
(b)
Annealing temperature should be optimized for each primer set based on the primer
melting temperature (T
m
). See section on primer design (p. 14).
(c)
The extension time should be at least 1 minute/kilobase of target. Typically, anything
smaller than 1kb uses a 1-minute extension, 2 minutes for >1kb, 3 minutes for >2kb, 4
minutes for >3kb, etc.
Comparison of Properties for Some Commonly-Used Thermostable DNA Polymerases.
Thermostable DNA Polymerase
Taq/ AmpliTaq Gold
Vent
Deep
Characteristic AmpliTaq
Platinum
Taq Tfl Tth (Tli) Vent
Pfu
>95% >95%
Resulting DNA ends 3 A 3 A 3 A 3 A Blunt Blunt Blunt
53 exonuclease activity Yes Yes Yes Yes No No No
35 exonuclease activity No No No No Yes Yes Yes
Want to explore the enzymology of Thermostable
DNA Polymerases more thoroughly?
Go to our online Polymerase Guide at:
www.promega.com/guides/
}
U
sing
a
P
roofre
ad
ing
Polym
e
rase
?
P
roofre
ad
ing
polym
e
rase
s
w
ork

a
little

slow
e
r
th
an
non-
proofre
ad
ing
polym
e
rase
s.
B
e

sure

to
inc
re
ase

th
e

e
xte
nsion
tim
e

to
at
le
ast
2
m
inute
s
pe
r
k
ilobase
.
A
lso,
you
m
ay
ne
e
d

2
-
3

m
ore

c
yc
le
s.
Optimization of PCR
Magnesium Concentration
Magnesium concentration is an important factor to
optimize when performing PCR. The optimal Mg
2+
concentration varies depending on the primers,
template, DNA polymerase, dNTP concentration and
other factors. Taq DNA polymerase is the most common
polymerase used for PCR. Taq has an optimal Mg
2+
range of 14mM MgCl
2
. Other polymerases may have
different optimal ranges. For example, Tth DNA
polymerase has a narrower optimal range (1.52.5mM
MgCl
2
), Tli DNA polymerase displays optimal activity at
26mM MgCl
2
, and Pfu DNA polymerase has an optimal
range of 26mM MgSO
4
. Tfl DNA polymerase has an
optimal range of 14mM Mg
2+
but performs better with
MgSO
4
than with MgCl
2
.
When using a pair of PCR primers for the first time, it is
advisable to perform a magnesium titration in 0.5 or
1.0mM increments to determine the optimal Mg
2+
concentration. Some primers will amplify equally well at
a number of Mg
2+
concentrations, while others may
have very specific Mg
2+
concentration requirements.
With too little Mg
2+
, the polymerase will have poor
activity. With too much Mg
2+
, nonspecific amplification
can become a problem. Nonspecific PCR products can
appear as a smear on a gel or as distinct bands of
inappropriate size. Too much Mg
2+
can also reduce the
fidelity of the DNA polymerase and lead to a higher
error rate.
Taq DNA polymerase is commonly supplied with buffers
containing a fixed concentration of Mg
2+
(giving a final
concentration of 1.5mM in the final reaction). Most Taq
DNA polymerase amplifications work well at this Mg
2+
concentration, and the reaction can still be optimized by
adding more Mg
2+
. Pfu DNA polymerase does not have
as great a dependence upon Mg
2+
and is most often
supplied with a buffer containing a final concentration of
2mM Mg
2+
. However, this does not mean that
optimization is unnecessary, and the final concentration
of Mg
2+
can be adjusted up to 6mM as needed.
Primer Design
PCR primers generally range from 1530 bases long
and are designed to flank the DNA region of interest.
Primers should have 4060% GC content, and care
should be taken to avoid sequences that might produce
intermolecular or intramolecular secondary structure. To
avoid the production of primer-dimers, the 3-ends of
the primers should not be complementary. Primer-
dimers unnecessarily sequester primers away from the
reaction and result in an unwanted polymerase reaction
that competes with the desired PCR product. Avoid
three G or C nucleotides in a row near the 3-end of the
primer, as this may result in nonspecific primer
annealing, increasing the synthesis of undesirable
products. Ideally, both primers should have nearly
identical melting temperatures (T
m
), allowing both
primers to anneal roughly at the same temperature. The
annealing temperature of the reaction is dependent upon
the primer with the lowest melting temperature. For
assistance with calculating the T
m
of any primer, a T
m
calculator is provided on the Promega web site at:
www.promega.com/biomath
14
Amplifying DNA
Vortex all MgCl
2
-containing solutions
thoroughly prior to use!
MgCl
2
solutions can form a
concentration gradient upon thawing.
Failure to vortex is a common source
of PCR failure!
N
e
e
d

to

c
a
lc
ula
te

y
o
u
r

T
m
?
G
o
to
P
rom
e
g
a's
B
ioM
at
h

pag
e

at
:
w
w
w
.prom
e
g
a.c
om
/biom
at
h
T
h
e

p
r
o
g
r
a
m

r
e
t
u
r
n
s

r
e
s
ult
s
f
r
o
m

t
h
r
e
e

d
if
f
e
r
e
n
t
p
u
b
lis
h
e
d

m
e
t
h
o
d
s

f
o
r

T
m
c
a
lc
ula
t
io
n
.
Y
o
u

c
a
n

a
ls
o

se
le
c
t
P
r
o
m
e
g
a

p
r
im
e
r
s

t
o

e
xa
m
in
e
t
h
e
ir

T
m
a
n
d

d
e
t
e
r
m
in
e

t
h
e
T
m
o
f

y
o
u
r

o
w
n

p
r
im
e
r
s

in
d
if
f
e
r
e
n
t

r
e
a
c
t
io
n

b
uf
f
e
r
s
.
15
Amplifying DNA
Optimization of PCR (continued)
Annealing Temperature
Annealing temperature is another factor that may need
to be optimized in PCR. The melting temperature (T
m
) of
the PCR primers should be in the range 4265C,
unless the primers fall into a special class, such as
degenerate primers, which have lower T
m
. Typically the
optimal annealing temperature is 5C of the primer
with the lowest T
m
. Ideally the T
m
of both primers will be
similar so that the optimal annealing temperatures are
close. If the melting temperatures are more than a few
degrees apart, one primer may need to be redesigned so
that the T
m
is closer to that of the other primer. A good
starting point is to set the annealing temperature equal
to the T
m
of the primers. If nonspecific amplification
occurs, this is a good indication that the annealing
temperature needs to be raised a few degrees. If the
PCR reaction yields no product, this may indicate that
the annealing temperature is too high and should be
reduced by several degrees.
Template Quantity
The amount of template required for successful
amplification is dependent upon the complexity of the
DNA sample. For example, in a 4kb plasmid containing a
1kb insert, 25% of the input DNA is the target of
interest. Conversely, a 1kb gene in the human genome
(3.3 10
9
bp) represents approximately 0.00003% of
the input DNA. Approximately 1,000,000-fold more
human genomic DNA is required to maintain the same
number of target copies per reaction. Two common
mistakes are the use of too much plasmid DNA or too
little genomic DNA. If possible, start with up to 10
4
copies of the target sequence to obtain a signal in
2530 cycles, but do not exceed 10ng/l (i.e.,
500ng/50l reaction).
Template Quality
The purity and integrity of the DNA template can also be
critical. Obviously there are numerous inhibitors that can
interfere with amplification. These may be copurified
from the original source of the nucleic acid (e.g., the
tissue from which the DNA was isolated). Contaminants
can also be introduced during the purification process.
Examples of common contaminants that can inhibit PCR
are phenol, ethanol, as little as 0.01% SDS or other
detergents, heparin and salts. These contaminants can
usually be removed by a simple phenol:chloroform
extraction followed by ethanol precipitation, or by use of
a PCR clean-up system (see Chapter 3). Some sample
types, such as blood, soil, fungus, plants with high
phenolic content, and fecal samples, are problematic
because they contain strong PCR inhibitors that can be
copurified with the DNA. An easy way to identify
inhibitors in your template nucleic acid is to add an
aliquot of template to the positive control reaction. If
this spiked control reaction fails, the template needs to
be further purified before amplification.
Test template quality by adding a control template that
you know amplifies easily and reliably. Combine your
problematic template with 1001,000 copies of the
control template, and amplify the control template.
Perform the same amplification with the control
template alone. If amplification of the control template
fails only when the problematic template is present,
inhibitors in the problematic template may be to blame.
R
e
ac
tion
anne
aling
te
m
pe
rature

sh
ould

be
5
C

of
th
e

T
m
of
th
e

P
C
R

prim
e
r
th
at
h
as
th
e

low
e
st
T
m
.
H
ow
M
any M
ole
c
ule
s in Your
D
N
A
T
e
m
plate
?
1g
of 1k
b d
sD
N
A
=

9
.12
x 10
11
m
ole
c
ule
s.
1g
of pG
E
M
V
e
c
tor D
N
A
=

2
.8
5
x 10
11
m
ole
c
ule
s.
1g
of lam
bd
a D
N
A
=

1.9
x 10
10
m
ole
c
ule
s.
1g
of E
. c
oli g
e
nom
ic
D
N
A
=

2
x 10
8
m
ole
c
ule
s.
1g
of h
um
an g
e
nom
ic
D
N
A
=
3
.0
4
x 10
5
m
ole
c
ule
s.
16
Amplifying DNA
PCR Enhancers
In some cases it may be helpful to add certain enhancing
agents to a PCR, despite all other attempts to optimize
conditions. Two good examples are the amplification of
GC-rich templates and amplification of templates that
form strong secondary structures, which can cause DNA
polymerases to stall. GC-rich templates can be
problematic due to inefficient separation of the two DNA
strands or because of the tendency of GC-rich primers to
form intermolecular and intramolecular secondary
structures that compete with template annealing. There
are many PCR-enhancing agents that act through a
number of different mechanisms. PCR-enhancing
reagents will not work with all reactions; the beneficial
effects are often template- and primer-specific.
Betaine, DMSO and formamide can be helpful when
amplifying GC-rich templates. Betaine reduces the
amount of energy required to separate the strands of a
GC-rich DNA template (1). Dimethylsulfoxide (DMSO)
and formamide are thought to aid in the amplification of
GC-rich templates in a similar manner by interfering
with the formation of hydrogen bonds between the two
strands of DNA (2). Some reactions that amplify poorly
in the absence of enhancers will give a strong PCR
product when betaine (1M), DMSO (110%), or
formamide (15%) are added to the reaction. DMSO
concentrations greater than 10%, and formamide
concentrations greater than 5% will cause inhibition of
Taq DNA polymerase and, presumably, other DNA
polymerases as well. (3).
In some cases, general stabilizing agents such as BSA
(0.1mg/ml), gelatin (0.11.0%), and nonionic detergents
(00.5%) can overcome failure to amplify a region of
DNA. These additives can increase the stability of the
DNA polymerase and may also coat the sides of the PCR
tubes so that reagents are not lost through adsorption
to the tube walls. BSA has also been shown to
overcome the inhibitory effects of melanin on RT-PCR
(4). Nonionic detergents, such as Tween
-20, NP-40,
and Triton
X-100, have the additional benefit of being

able to overcome the inhibitory effects of trace amounts
of strong ionic detergents, such as 0.01% SDS (5).
Ammonium ions can make a PCR reaction more tolerant
of nonoptimal conditions. For this reason, some PCR
reagents include 1020mM (NH
4
)
2
SO
4
. Other PCR
enhancers include glycerol (520%), polyethylene glycol
(515%), and tetramethyl ammonium chloride (TMAC;
60mM). The effects of these enhancers are very
template- and primer-specific. It may be easier to design
new primers and determine the optimal conditions for
the new primer pair than to do multiple experiments
with some of these less useful enhancers.
References
1. Rees, W., Yager, T.D., Korte, J. and von Hippel, P.H. (1993) Betaine can
eliminate the base pair composition dependence of DNA melting.
Biochemistry 32,13744.
2. Geiduschek, E.P. and Herskovitz, T.T. (1961) Nonaqueous solutions of DNA.
Reversible and irreversible denaturation in methanol. Arch. Biochem.
Biophys. 95, 11429.
3. Varadaraj, K. and Skinner, D. (1994) Denaturants or cosolvents improve the
specificity of PCR amplification of a GC-rich DNA using genetically
engineered DNA polymerases. Gene 140, 15.
4. Giambernardi, T.A., Rodeck, U. and Klebe, R.J. (1998) Bovine serum
albumin reverses inhibition of RT-PCR by melanin. BioTechniques 25,
56466.
5. Gelfand, D.H. and White, T.J. (1990) Thermostable DNA polymerase. In:
PCR Protocols: A Guide to Methods and Applications. Innis, M.A., Gelfang,
D.H., Sninsky, J.J., and T.J. White (eds.) Academic Press, San Diego, CA.
pp. 12941.
bp
1,198
350
222
Colorless
GoTaq Buffer
Green
GoTaq Buffer
3
8
2
5
T
A
0
8
_
2
A
N
o
n
e
D
M
S
O
b
e
t
a
i
n
e
b
e
t
a
i
n
e
D
M
S
O

+

b
e
t
a
i
n
e
D
M
S
O

+

b
e
t
a
i
n
e
N
o

t
e
m
p
l
a
t
e
N
o
n
e
D
M
S
O
N
o

t
e
m
p
l
a
t
e
Amplification of a fragment of the human retinoblastoma gene using GoTaq
DNA Polymerase with Colorless GoTaq Reaction Buffer or Green GoTaq
Reaction Buffer with and without the addition of enhancing agents DMSO and
betaine. Amplification reactions contained 500ng human genomic DNA, 0.8M of each
primer and 1.25u GoTaq DNA Polymerase in a final volume of 50l. Reactions contained
no additives, 5% DMSO, 1M betaine or 5% DMSO + 1M betaine as indicated. No-template
control reactions were included. Amplification primers and cycling conditions are as
published in Frackman, S. et al. (1998) Betaine and DMSO: Enhancing agents for PCR.
Promega Notes 65, 2729.
Troubleshooting
Most troubleshooting of PCR involves running through
the possible areas of optimization. Promega has an
extensive PCR troubleshooting guide included in the
PCR Core Systems Technical Bulletin #TB254.
Also, Promega has an interactive troubleshooting and
optimization tool available online called the
Amplification Assistant
SM
. You simply input your
reaction parameters and your results, and the
Amplification Assistant
SM
analyzes your situation and
returns possible solutions to your problems.
17
Amplifying DNA
RT-PCR
Looking for information, tips and techniques for
RT-PCR? Request the free RNA Analysis Notebook.
Ask for literature #BR120 from your local Promega
distributor or Promega Representative. Also available
online at: www.promega.com/guides/
H
ave
m
ore
que
stions?
C
ontac
t P
rom
e
g
a
T
e
c
h
nic
al S
e
rvic
e
s:
te
c
h
se
rv@
prom
e
g
a.c
om
S
c
ie
ntists se
rving
sc
ie
ntists.
Need a guide to general PCR
optimization and troubleshooting?
Get PCR Core Systems Technical
Bulletin #TB254 online at:
or request a printed copy from your
local Promega representative.
Inte
r
ac
tiv
e

P
C
R

t
rouble
sh
ootin
g
and

optim
iz
ation
tool
av
ailable

at
:
w
w
w
.prom
e
g
a.c
om
/am
plific
ationasst/
D
e
sig
ne
d

for
routine
P
C
R

and

R
T
-
P
C
R
.
18
Amplifying DNA
Routine PCR
PCR Master Mix: Robust, Convenient Amplification
Promegas PCR Master Mix
(f,g)
is designed for the rapid
and convenient amplification of many common genomic
and cDNA templates (1). PCR Master Mix, formulated as
a 2X solution, offers a single-tube format for PCR setup,
reducing pipetting times, steps and errors as well as
greatly reducing reagent waste. All necessary PCR
components, except for primers and template DNA, are
contained in the Master Mix. Stability is also a key
feature of PCR Master Mix, which can be stored for up to
24 months at 4C or be put through as many as 20
freeze-thaw cycles without loss of performance. PCR
Master Mix provides 1.25u of Taq DNA Polymerase,
Reaction Buffer, 200M of each dNTP, and 1.5mM Mg
2+
in the final reaction.
Reference
1. Denhart, M. and Doraiswamy, V. (2001) Performance advantages designed
into Promegas PCR Master Mix. Promega Notes 78, 912.
M M
3
0
6
3
T
A
0
9
_
0
A
1,000 100 10 2 0
Template Copies per Reaction
3
9
8
6
M
A
0
2
_
3
A
PCR Master Mix
Reaction
Buffer
dNTPs
Primer
Template
Mg
2+
Taq DNA
Polymerase
Typical Reaction Set-Up with PCR Master Mix.
Template (up to 10
4
copies of target) Xl
Primers (50pmol each or 1M final conc.) Yl
Nuclease-Free Water (provided) Zl
PCR Master Mix* 25l
Total Volume 50l
* Provides dNTPs (200M each), Mg
2+
(1.5mM), and Taq DNA Polymerase (1.25u) at the
final 1X concentration.
Detection of low copy number templates using PCR Master MIx. A 360bp
portion of the single-copy 1-antitrypsin gene was amplified from the indicated amounts of
Human Genomic DNA (Cat.# G3041). Lane M, 100bp DNA Ladder (Cat.# G2101).
Scalability of PCR Master Mix. The 360bp 1-antitrypsin message was amplified from
10 or 100 copies of Human Genomic DNA (Cat.# G3041) in 10, 25 and 50l reaction
volumes as indicated.
PCR Master Mix in two-step RT-PCR. Amplification of a 533bp portion of the
caspase-3 cDNA from 20l reverse transcription reaction. Reverse transcription was
performed using the ImProm-II Reverse Transcription System (Cat.# A3800) and the
indicated amount of total RNA. The cDNA in the entire 20l reaction was amplified by
adding 15l of PCR Master Mix, 2l of gene-specific primers and 13l of Nuclease-Free
Water (Cat.# P1195). Further details of the experiment may be found in Miller, K., Moravec,
R. and Riss, T. (2001) An integrated approach to studying apoptosis: From gene expression
to cellular events. Cell Notes 2, 46.
M
10l 25l 50l
Reaction Volume
3
2
7
8
T
A
0
3
_
1
A
10 100 10 100 10 100
360bp
1
0
0
n
g
1
0
n
g
1
n
g
1
0
0
p
g
1
0
p
g
1
p
g
3
4
4
1
T
A
0
6
_
1
B
PCR Master Mix
Cat.#: M7501 (10 reactions)
M7502 (100 reactions)
M7505 (1,000 reactions)
Protocol:
www.promega.com/tbs/9pim750/9pim750.html
Citations detailing use of PCR Master Mix online at:
Cell Notes and
Promega Notes
are available online at:
www.promega.com
or upon request from
Promega.
19
Amplifying DNA
Routine PCR (continued)
GoTaq DNA Polymerase:
Direct-to-Gel Amplification
GoTaq DNA Polymerase
(e,g)
contains native Taq DNA
Polymerase in a proprietary formulation. The GoTaq
enzyme is supplied with 5X Green and 5X Colorless
GoTaq Reaction Buffers. The Green Reaction Buffer
contains a compound that increases sample density so
that samples sink easily into the wells of an agarose gel.
The Green Reaction Buffer also contains two dyes (a blue
dye and a yellow dye) that separate during electrophoresis
and can be used to monitor migration progress. This
allows reactions to be directly loaded onto agarose gels
without the need for loading dye. The blue dye migrates at
the same rate as 35kb DNA fragments in a 1% agarose
gel. The yellow dye migrates at a rate faster than primers
(<50bp) in a 1% agarose gel. The Colorless GoTaq
Reaction Buffer has the same formulation as the Green
Reaction Buffer but does not contain dyes. The Colorless
Reaction Buffer is recommended for any application where
absorbance or fluorescence measurements of the PCR
amplimer are necessary before clean-up. Both 5X buffers
are supplied at pH 8.5 and contain MgCl
2
at a
concentration of 7.5mM, giving a final concentration of
1.5mM in the reaction.
Compatibility of GoTaq DNA Polymerase with Upstream and Downstream Applications.
Product Cat.# Green Reaction Buffer Colorless Reaction Buffer
T-Vector Cloning
pGEM
-T & pGEM
-T Easy Systems
(h,i)
A1360, A1380, A3600, A3610 Yes Yes
pTARGET Mammalian Expression Vector System
(i,j)
A1410 Yes Yes
PCR Clean-Up
Wizard

(a)
A1930 Yes Yes
Wizard
SV 96 PCR Clean-Up System A9340 Yes Yes

Wizard

(k)
A9281 Yes Yes
Wizard

(l)
A7170 Yes Yes
Two-Step RT-PCR
Reverse Transcription System
(m,n)
A3500 Yes Yes
ImProm-II Reverse Transcription System
(m,n)
A3800 Yes Yes
Transcription/Translation
TNT
T7 Quick for PCR DNA

(m,n,o)
L5540 Yes Yes
Separation of the components of the GoTaq Green Reaction Buffer during
electrophoresis. PCR samples amplified using GoTaq DNA Polymerase and GoTaq Green
Reaction Buffer were loaded onto an agarose gel. Samples are shown before (A) and after (B)
electrophoresis. Volumes indicate the amount of amplification reaction loaded on the gel.
Amplification of various templates with GoTaq DNA Polymerase and other
Promega Taq DNA Polymerase formulations. The reactions for each template are
loaded in this order: Taq DNA Polymerase in Storage Buffer B (Cat.# M1661); Taq DNA
Polymerase in Storage Buffer A (Cat.# M1861); GoTaq DNA Polymerase in Colorless
Reaction Buffer; GoTaq DNA Polymerase in Green Reaction Buffer. Reactions without the
Green GoTaq Reaction Buffer require the addition of loading dye prior to electrophoresis.
Cat.#: M3001 (100u; 80 reactions)
M3005 (500u; 400 reactions)
M3008 (2,500u; 2,000 reactions)
Supplied with enzyme (5u/l), 5X Green GoTaq
Reaction Buffer and 5X Colorless GoTaq Reaction
Buffer. Sufficient to give the indicated number of 50l
reactions using 1.25u of enzyme per reaction.
Protocol:
bp
4,000
2,000
1,500
1,000
500
250
3,000
360bp 1.1kb 1.8kb 2.4kb 3.1kb
M M
3
8
2
4
T
A
0
8
_
2
A
2 3 4 5 6 7 8 9 10 11 12 13 14 1516 1718 19 20 1
20
Amplifying DNA
Routine PCR (continued)
Taq DNA Polymerase & PCR Core Systems:
Value and Quality
Promega is a premier supplier of native Taq DNA
Polymerase. We offer many options for your needs. You
can assemble your own reagents from separate Taq
DNA Polymerase and dNTPs, or purchase the PCR Core
Systems to get everything together in one package. The
PCR Core Systems
(f)
are supplied with a Technical
Bulletin that contains thorough coverage of
considerations involved in routine PCR amplification and
extensive troubleshooting information.
Taq DNA Polymerase in Storage Buffer A & B
Promega first offered Taq DNA Polymerase stabilized
with Triton
X-100 (Taq DNA Polymerase in Storage

Buffer A). Later we developed Taq DNA Polymerase
stabilized with Tween
-20 and NP-40 (Taq DNA

Polymerase in Storage Buffer B). In most cases there is
no difference in performance. One key distinction
between the two products is compatibility with other
suppliers reaction buffers. Taq DNA Polymerase in
Storage Buffer A must be used with the supplied
Reaction Buffer, which contains 0.1% Triton
X-100 at
the 1X concentration. Taq DNA Polymerase in Storage
Buffer B does not have this requirement and can be
used either with the supplied Promega Reaction Buffer
or with other Taq DNA polymerase reaction buffers.
PCR Core System I
Cat.#: M7660 (200 50l reactions; 1.25u Taq DNA
Polymerase/reaction)
Comes with 250u Taq DNA Polymerase in Storage
Buffer B, Taq DNA Polymerase 10X Reaction Buffer
without MgCl
2
, Taq DNA Polymerase 10X Reaction
Buffer with MgCl
2
(1.5mM at 1X), 25mM MgCl
2
, PCR
Nucleotide Mix.
PCR Core System II
Cat.#: M7665 (200 reactions; 1.25u Taq
Polymerase/50l reaction)
Same components as M7660 plus Positive Control
Plasmid DNA template and Upstream and Downstream
Control Primers.
Protocol:
Taq DNA Polymerase in Storage Buffer A
M1868 (2,500u; 2,000 reactions)
Supplied with Taq DNA Polymerase 10X Reaction
Buffer, 25mM MgCl
2
. One reaction uses 1.25u of
enzyme.
Taq DNA Polymerase in Storage Buffer B
M1668 (2,500u; 2,000 reactions)
Supplied with Taq DNA Polymerase 10X Reaction
Buffer, 25mM MgCl
2
. One reaction uses 1.25u of
enzyme.
Citations for use of Taq DNA Polymerase online at:
3
9
8
7
M
A
0
2
_
3
A
Primers
Template
All components
supplied in
PCR Core Systems
Taq DNA
Polymerase
dNTPs
10X Thermophilic
Reaction Buffer
MgCl
2
T
h
e

P
C
R

C
o
r
e
S
y
s
t
e
m
s

a
r
e

g
r
e
a
t

f
o
r
r
e
s
e
a
r
c
h
e
r
s

ju
s
t le
a
r
n
in
g
P
C
R
See p. 12 for
typical reaction
set-up.
S
e
e

p
.
2
3

f
o
r

d
N
T
P
s
.
21
Amplifying DNA
Incorporation fidelity can be an important consideration
for cloning projects. Thermostable enzymes with a
35 exonuclease activity, commonly known as
proofreading activity, offer the highest fidelity in
amplification reactions. Proofreading enzymes like Pfu
and Tli DNA Polymerase
(e)
offer three- to sixfold higher
fidelity than standard Taq DNA Polymerase. In general,
proofreading enzymes extend primers a little slower
than Taq DNA Polymerase and thus typically require
longer extension times and a few more cycles. Assume
2 minutes per kilobase of amplimer, and add 23 cycles
to your reaction when using a proofreading enzyme.
With Pfu DNA Polymerase
(e)
, it is important to use the
Reaction Buffer supplied with the enzyme for maximum
fidelity. Pfu Reaction Buffer is formulated to give
maximum fidelity, not maximum yield. After all, you use
Pfu for fidelity not yield. If you need greater yield, use
more template DNA.
Reference
1. Cline, J., Braman, J.C. and Hogrefe, H.H. (1996) PCR fidelity of Pfu DNA
polymerase and other thermostable DNA polymerases. Nucl. Acids Res. 24,
354651.
Comparison of sources of native Pfu DNA Polymerase. A 1.2kb fragment of human
1-antitrypsin gene was amplified using Pfu DNA Polymerase from Promega (Panel A) and
from another supplier (Panel B). The target was amplified from decreasing amounts of Human
Genomic DNA (Cat.# G3041) as indicated. Lane M, 100bp DNA Ladder (Cat.# G2101).
Accuracy of thermostable polymerases. The accuracy of Pfu DNA Polymerase has
been reported by Cline et al. as 7.7 10
5
. Using the PCR-based forward mutation assay,
they reported the accuracy of Pfu as approximately twofold higher than Vent
(Tli DNA
Polymerase) and approximately sixfold higher than Taq DNA Polymerase (1).
Pfu DNA Polymerase*
Each provided with enzyme (5u/l), Pfu DNA
Polymerase 10X Reaction Buffer (2mM MgSO
4
@ 1X)
sufficient to give the indicated number of 50l
reactions using 1.25u of enzyme.
Protocol:
Citations for use of Pfu DNA Polymerase online at:
*Not available in North America.
Tli DNA Polymerase
Supplied with enzyme (5u/l), Thermophilic DNA
Polymerase 10X Reaction Buffer and 25mM MgCl
2
.
Sufficient to give the indicated number of 50l
reactions using 1.25u of enzyme/reaction.
Citations for use of Tli DNA Polymerase online at:
3
9
7
3
M
A
0
2
_
3
A
Pfu Tli Taq
0
1
2
3
4
5
6
7
8
A
c
c
u
r
a
c
y

1
0
5
DNA Polymerase
M 30 3 0.3 0.03 0.0
Promega
M 30 3 0.3 0.03 0.0
Supplier A
bp
1,500
1,000
500
100
1.2kb
product
2
3
5
5
T
A
0
8
_
8
A
A. B.
Proofreaders have a 3
5
exonuclease activity that is lacking
in
non-proofreading
enzymes like Taq
DNA Polymerase. Proofreading
enzymes
can degrade primers if the reaction is
allowed to sit for too long
prior to
amplification. We recommend setting
up
reactions on ice and adding
the
proofreading
polymerase just prior to
placing
the reaction into a preheated
thermal cycler.
U
sag
e
H
int: Inc
re
ase
am
plific
ation e
xte
nsion
tim
e
s 2
X
ove
r stand
ard
T
aq D
N
A
Polym
e
rase

(e.g
., 2
m
in/k
b)
and
ad
d
2
3
c
yc
le
s.
22
Amplifying DNA
Hot Start Methodology
Hot start PCR is a commonly used technique to reduce
nonspecific amplification. One cause of nonspecific
amplification is the assembly of PCR reactions at room
temperature or on ice. Under these conditions, the PCR
primers may be able to anneal to various non-
complementary positions on the template. Although
activity of thermostable DNA polymerases at room
temperature or 4C is usually less than 25%, they can
extend nonspecifically annealed primers at these
temperatures. Any newly synthesized product is
completely complementary to the PCR primer, allowing
the primer to anneal specifically to this region during
PCR, resulting in an undesired amplification product.
Hot start PCR can also reduce the amount of primer-
dimer formed. Primer-dimers result from
complementarity between the 3 ends of the PCR primers.
At room temperature or on ice, these complementary
regions anneal and the polymerase extends the ends to
produce a primer-dimer. Primer-dimers often appear as a
diffuse band at ~50100bp on ethidium bromide-stained
gels. Both nonspecific products and primer-dimers can
compete with the desired amplification reaction for
reagents. By avoiding the conditions that lead to
nonspecific amplification, hot start PCR can improve the
yield of the desired PCR product.
There are several ways to perform hot start PCR. The
reaction can be assembled on ice or at room
temperature, omitting the DNA polymerase until the
reaction has been placed in the thermal cycler and
heated to 6065C. Once the reaction has reached
6065C the desired amount of polymerase can be
added. This prevents the polymerase from extending
primers until the higher temperature is reached and
primer annealing is more specific. The method is quite
effective but can be labor-intensive, particularly if
dozens of amplification reactions are involved.
Another approach to hot start PCR involves the use of
wax to physically sequester one or more critical reaction
components until the appropriate temperature is
reached. Wax beads can be added to a PCR before the
addition of the DNA polymerase. Heating the PCR to
60C melts the wax, which forms a liquid layer over the
surface of the reaction, eliminating the need for mineral
oil. Upon cooling to 4C, the wax solidifies. The DNA
polymerase is added onto this wax layer and, as the
PCR is heated during the first denaturation step, the wax
melts and the polymerase can access the other PCR
reagents. This method is labor intensiverequiring an
additional heating and cooling step to prepare the wax
layer. Also, opening the PCR tube to add the polymerase
increases the risk of contamination. Additionally, the
solid wax layer that forms upon cooling to 4C will clog
pipet tips when attempting to break through the wax to
pipet the PCR. Thus, it is often necessary to use one
pipet tip to puncture the wax layer and a second pipet
tip to remove the PCR products.
TaqBead Hot Start Polymerase
Enter Promegas TaqBead Hot Start Polymerase
(f)
. By
impregnating a wax bead with Taq DNA Polymerase, the
additional heating and cooling steps to form the wax
layer are eliminated. A single bead is added to each 50l
reaction, and as the reaction is heated, the wax melts
and releases the polymerase. The molten wax rises to
the surface of the PCR where it forms an incomplete
barrier. In thermal cyclers with heated lids, a hole
remains above the reaction to ease pipetting. To prevent
evaporation in thermal cyclers without heated lids, we
recommend adding mineral oil to each PCR. The molten
wax and mineral oil will mix during the thermal cycling
to form a single layer, which solidifies when the reaction
is cooled to 4C and can clog pipet tips.
Cat.#: M5661 (100 reactions)
Supplied with 100 beads (1.25u Taq DNA Polymerase
in Storage Buffer B/bead), Thermophilic DNA
Polymerase 10X Reaction Buffer, and 25mM MgCl
2
.
Protocol:
Citations for use of TaqBead Polymerase online at:
Hot start amplification reduces the yield of nonspecific amplification products.
Aliquots of 10, 1 or 0.1pg pGEM
-luc Vector
(h,p)
(Cat.# E1541) were diluted in 30ng of
Human Genomic DNA (Cat.# G3041). A 1.8kb luciferase gene product was amplified by
PCR using Taq DNA Polymerase (Storage Buffer B) or TaqBead Hot Start Polymerase in
Promega Reaction Buffer supplemented with 2mM MgCl
2
. Details are provided in Miller, K.,
Smith, R. and Storts, D. (1996) Improved PCR amplification using TaqBead Hot Start
Polymerase. Promega Notes 60, 26.
bp
2,645
1,605
1,198
676
517
396
primer-
dimer
1.8kb
M 10 1 0.1 10 1 0.1
Taq
Cold Start
TaqBead
Hot Start
pg of
target
1
5
8
6
T
A
0
9
_
6
A
23
Amplifying DNA
dNTPs
Promega is a premier supplier of high-quality dNTPs in
bulk form or premixed in the PCR Nucleotide Mix.
Promegas dNTPs are >99% triphosphate with verified
concentrations. All dNTPs, whether bulk or premixed,
are DNase- and RNase-free, and are functionally tested
in amplification reactions. The PCR Nucleotide Mix is
also functionally tested in RT-PCR.
PCR Nucleotide Mix
Cat.#: C1141 (200l; 200 reactions)
C1145 (1,000l; 1,000 reactions)
The PCR Nucleotide Mix supplies a single solution
containing each dNTP (dATP, dTTP, dGTP, dCTP) at
10mM. Reaction size is considered to be 200M of
each dNTP in a 50l reaction. Each reaction uses 1l
of PCR Nucleotide Mix.
Custom and bulk PCR Nucleotide Mix sizes are
available.
Protocol:
www.promega.com/tbs/9pic114/9pic114.html
Set of dATP, dCTP, dGTP and dTTP
Cat.#: U1330 (10mol each; 1,000 reactions)
U1240 (40mol each; 4,000 reactions)
Set of dUTP, dATP, dCTP and dGTP
Cat.#: U1335 (10mol each; 1,000 reactions)
Each dNTP is supplied at 100mM. Reaction size is
considered to be 200M each dNTP in a 50l PCR
reaction.
Custom and bulk dNTP sizes are available.
RT-PCR functional assay using PCR Nucleotide Mix. The dNTPs were used following
50 freeze-thaw cycles. Amounts of template RNA: lane 1, 25fmol; lane 2, 2.5fmol; lane 3,
250amol; lane 4, 25amol; lane 5, 2.5amol; lane 6, 250zmol; lane 7, no template control.
Stability of dNTPs. The integrity of the RT-PCR products on the gel demonstrates the
stability of the dNTPs after storage under the conditions listed. The dNTPs used in each RT-
PCR were stored as follows prior to use: Lane 2, 1 freeze-thaw cycle; lane 3, 50 freeze-thaw
cycles; lane 4, 1 year at 20C; lane 5, negative PCR control; lanes 1 and 6, 100bp DNA
Ladder (Cat.# G2101).
1 2 3 4 5 6
3
5
2
7
T
A
0
8
_
1
A
bp
1,500
1,000
500
1 2 3 4 5 6 7
3
5
2
8
T
A
0
8
_
1
A
24
Amplifying DNA
Routine PCR
Product Size Cat.#
PCR Master Mix
(f,g)
100 reactions M7502
1,000 reactions M7505
For Laboratory Use. A reaction consists of 25l of the 2X PCR Master Mix in a 50l total volume. Supplied with Nuclease-Free Water.
Product Size Cat.#
(e,g)
100u M3001
500u M3005
2,500u M3008
For Laboratory Use. Supplied with 5X Green GoTaq Reaction Buffer and 5X Colorless GoTaq Reaction Buffer. Both buffers contain 1.5mM Mg
2+
at the 1X concentration.
Product Size Cat.#
PCR Core System I
(f)
200 reactions M7660
PCR Core System II
(f)
200 reactions M7665
For Laboratory Use. PCR Core Systems provide Taq DNA Polymerase in Storage Buffer B, PCR Nucleotide Mix, Taq DNA Polymerase 10X Reaction Buffers with and without MgCl
2
, and 25mM MgCl
2
sufficient for 200 50l reactions containing 1.25u of Taq DNA Polymerase. PCR Core System II also contains Positive Control Plasmid DNA template, and Upstream and Downstream Control
Primers.
Product Size Cat.#
(e)
100u M1861
(Supplied with Taq DNA Polymerase 10X Reaction Buffer without MgCl
2
, and 25mM MgCl
2
.) 500u M1865
2,500u M1868
(e)
100u M2861
(Supplied with Taq DNA Polymerase 10X Reaction Buffer with MgCl
2
, giving 1.5mM Mg
2+
at the 1X concentration.) 500u M2865
2,500u M2868
(e)
100u M1661
(Supplied with Taq DNA Polymerase 10X Reaction Buffer without MgCl
2
, and 25mM MgCl
2
Solution.) 500u M1665
2,500u M1668
(e)
100u M2661
(Supplied with Taq DNA Polymerase 10X Reaction Buffer with MgCl
2
, giving 1.5mM Mg
2+
2,500u M2668
For Laboratory Use.
Product Size Cat.#
Pfu DNA Polymerase
(e)
* 100u M7741
(Supplied with Pfu DNA Polymerase 10X Reaction Buffer with MgSO
4
, giving 2mM Mg
2+
Tli DNA Polymerase
(e)**
(Supplied with Thermophilic DNA Polymerase 10X Reaction Buffer and 25mM MgCl
2
.) 50u M7101
*Not Available in North America. **For Laboratory Use.
25
Amplifying DNA
Hot Start Polymerase
Product Size Cat.#
(f)
(Supplied with Thermophilic DNA Polymerase 10X Reaction Buffer and 25mM MgCl
2
.) 100 reactions M5661
For Laboratory Use. One bead per reaction; 1.25u Taq DNA Polymerase per bead.
dNTPs
Product Size Cat.#
PCR Nucleotide Mix 200l C1141
(Contains 10mM each dNTP; use 1l per 50l reaction.) 1,000l C1145
Set of dATP, dCTP, dGTP, and dTTP 10mol U1330
(100mM each dNTP. Individual tubes available.) 40mol U1240
200mol U1410
Set of dUTP, dCTP, dGTP, and dATP
(q)
10mol U1335
(100mM each dNTP.) 40mol U1245
For Laboratory Use.
Accessories
Product Size Cat.#
Promega 10 Barrier Tips, 960/pk 0.510l A1491
Promega 10E Barrier Tips, 960/pk 0.510l A1501
Promega 10F Barrier Tips, 960/pk 0.510l A1511
Promega 20 Barrier Tips, 960/pk 220l A1521
Promega 1000 Barrier Tips, 480/pk 1001,000l A1561
Mineral Oil* 12ml DY1151
Nuclease-Free Water* 150ml P1195
*For Laboratory Use.
Tip and Pipette Compatibility Guide
Oxford
Size Pipetman
Eppendorf
Benchmate
Finnpipette
Promega 10 0.510l P-2; P-10 0.510l 0.510l Digital

Promega 10E 0.510l P-2; P-10 0.510l 0.510l
Promega 10F 0.510l 0.510l
Promega 20 220l P-20 220l
Promega 100 10100l P-100 10100l 1050l 540l
Promega 200 50200l P-200 EDP250l 40200l 40200l
Promega 1000 1001,000l P-1000 2001,000l 2001,000l
26
PCR Clean-Up
Overview
Downstream applications such as T-Vector cloning,
restriction digestion and direct sequencing benefit from
clean-up of PCR amplimers. T-vector cloning has a
tremendous dependence upon PCR product purity.
Although unpurified amplification reactions can be used
for T-vector cloning, more screening of the resulting
colonies is generally necessary to find the specific clone
of interest. This is because unpurified PCR amplification
reactions can contain primer-dimers and nonspecific
amplimers in much higher molar quantities than the PCR
product of interest. These nonspecific products compete
for ligation with the amplimer of interest. Typically, an
experiment with unpurified products will produce a large
number of colonies, many of which contain small,
nonspecific PCR products as inserts. Thus, the efficiency
of the cloning experiment is reduced. In one case the
percentage of colonies containing the correct insert was
67% using unpurified PCR products. In contrast, when
purified PCR products were used >90% of colonies
contained the correct insert (1).
Gel Isolation
Gel isolation is the most effective way to isolate the PCR
product you need for your downstream applications.
Agarose gel electophoresis allows you to separate the
desired amplimer from any nonspecific bands, primers
and primer-dimers. You visualize your gel quickly on a
UV lightbox, cut out the band of interest and then purify
the product. Gel isolation is typically used if the
downstream application is cloning and additional bands,
representing nonspecific amplimers or primer-dimer, are
present on the gel. The agarose is melted and combined
with a chaotrope like guanidine, and the DNA is then
bound to silica. Agarose and guanidine are quickly and
efficiently removed by an alcohol wash, and the purified
DNA is eluted in water.
Direct Isolation
Many downstream applications such as DNA sequencing
and single nucleotide polymorphism (SNP) analysis
require that salts, dNTPs, proteins and primers be
removed from the amplification product as they can
interfere with further enzymatic reactions. Many
researchers use a simple ethanol precipitation prior to
sequencing. This removes most dNTPs and salts but
leaves behind protein and may also leave primers.
Phenol:chloroform extraction can remove protein
contaminants but recovery rates can be low, and the use
of organics is undesirable. Use of silica technology
simplifies the whole process. The amplification reaction
products are bound to silica in the presence of a
chaotrope like guanidine. Salts, dNTPs and primers pass
by the silica. Primer-dimers and nonspecific amplimers
smaller than 70bp are not bound efficiently. A simple
alcohol wash removes the guanidine and protein while
material >70bp is retained. The purified DNA is eluted in
water or another low-ionic strength solution. The
success of the downstream application is dependent
upon the specificity of the amplification reaction, as any
nonspecific amplimers will be copurified with the
specific product of interest.
Reference
1. Buros, M. and Betz, N. (2002) Removal of ethidium bromide and calf
intestinal alkaline phosphatase using the Wizard
SV Gel and PCR Clean-

Up System. eNotes:
www.promega.com/enotes/applications/ap0045_tabs.htm
Gel Purification
4
0
2
0
M
A
0
3
_
3
A
PCR Reaction
Wizard
SV
Gel & PCR
Clean-Up System
Wizard
PCR
Preps DNA
Purification
System
Wizard
SV 96
PCR Clean-Up
System
Wizard
MagneSil PCR
Clean-Up System
Direct Purification
27
PCR Clean-Up
Wizard
SV Gel and PCR

Clean-Up System
The Wizard

(k)
is
designed to extract and purify DNA fragments directly
from PCR reactions or from agarose gels. Fragments of
100bp10kb can be recovered from standard or low-
melt agarose gels in either Tris acetate (TAE) or Tris
borate (TBE) buffer. Up to 95% recovery is achieved,
depending upon the DNA fragment size. This
membrane-based system, which can bind up to 40g of
DNA, allows recovery of isolated DNA fragments or PCR
products in as little as 15 minutes, depending on the
number of samples processed and the protocol used.
Samples can be eluted in as little as 15l of Nuclease-
Free Water. The purified DNA can be used for automated
fluorescent sequencing, cloning, labeling, restriction
enzyme digestion or in vitro transcription/translation
without further manipulation.
Gel analysis of PCR products before and after gel extraction using the
Wizard
SV Gel and PCR Clean-Up System. Recovery of various sizes of unpurified

(U) and purified (P) PCR products. Purified products were extracted from a 1% agarose gel
run with TAE buffer. Lane M, 1kb DNA Ladder (Cat.# G5711).
U
U: Unpurified P: Purified
100bp 500bp 1,000bp 3,199bp
M
3,000bp
1,000bp
P U P U P U P
3
7
8
9
T
A
0
7
_
2
A
3
7
9
0
T
B
0
8
_
2
A
U
100bp 200bp 500bp
P P P U P P P U P P P
U P P P U P P P
1,000bp 1,500bp
Gel analysis of PCR products before and after direct purification using the
Wizard
SV Gel and PCR Clean-Up System. DNA fragments of the sizes indicated
were analyzed before (U) and after (P) direct purification from amplification reactions.
Elution volume versus recovery for a 700bp PCR product purified directly from
an amplification reaction using the Wizard
SV Gel and PCR Clean-Up

System. One hundred percent represents recovery with 50l elution volume. Adapted from
Table 4 in Betz, N. and Strader, T. (2002) Clean up with Wizard
SV for Gel and PCR.

Wizard

Cat.#: A9281 (50 preps)
A9282 (250 preps)
Protocol:
Effect of Various PCR Additives on Recovery of a 1,000bp
PCR Product Using the Wizard
SV Gel and PCR Clean-Up

System Direct Purification Method.
Percent Recovery
PCR Additive Relative to No Additive
No Additive 100%
1M Betaine 94%
1M Q-Solution 97%
0.1% Triton
X-100 92%
0.1% Tween
20 87%
0.1% NP-40 82%
5% Glycerol 87%
5% Formamide 90%
5% DMSO 87%
0.5M Tetramethylene Sulfoxide 94%
0.4M Sulfolane 94%
0.4M 2-Pyrolidene 95%
1mM Tartrazine 100%
1% Ficoll
-400 100%
3
9
7
2
M
A
0
2
_
3
A
10 15 25 50 75 100
0
10
20
30
40
50
60
70
80
90
100
P
e
r
c
e
n
t

R
e
c
o
v
e
r
y
Elution Volume (l)
L
i
n
e
a
r

D
N
A

a
s

b
i
g
a
s

1
0
k
b

c
a
n

b
e
p
u
r
if
ie
d

w
i
t
h

u
p

t
o
9
5
%

r
e
c
o
v
e
r
y
.
28
PCR Clean-Up
Wizard
SV 96
PCR Clean-Up System
The Wizard
SV 96 PCR Clean-Up System provides a

fast, simple technique for the efficient isolation of
purified DNA fragments generated by PCR amplification.
Walkaway automation is easily achieved on any 96-well
liquid handling workstation equipped with a gripper and
vacuum apparatus. Double-stranded DNA fragments can
be purified from 96 samples in less than 20 minutes.
Purification is achieved without phenol:choroform
extraction or ethanol precipitation. Optimized methods
are available for the Beckman Biomek
2000 and
FX instruments.
Microarray of purified PCR products. Representative microarray blocks of PCR
product purified using the Wizard
SV 96 PCR Clean-Up System and hybridized to

complementary Cy
3-labeled cDNA. PCR DNA was isolated from a standard amplification

reaction or from a reaction containing 1M betaine. No effect of betaine is observed.
Agarose gel analysis of PCR fragments purified on the Biomek
2000 liquid
handler. PCR fragments of 100, 200, 300, 500 and 1,000bp were purified using the
Wizard
SV 96 PCR Clean-Up System on the Biomek
2000 robotic workstation. Both

purified (P) and unpurified (U) fragments were separated on an ethidium bromide-stained
2% agarose gel. Percent recovery ranged from 71 3% for the 100bp fragment to 100
1% for the 1,000bp fragment. Lane M, 100bp DNA Ladder (Cat.# G2101).
Wizard
SV 96 PCR Clean-Up System

Cat.#: A9340 (1 96 preps)
A9341 (4 96 preps)
A9342 (8 96 preps)
Protocol:
For information on automated methods, visit:
1M Betaine Standard PCR
3
7
9
6
T
B
0
9
_
2
A
100bp 200bp 300bp 500bp 1,000bp
3
8
0
1
T
A
0
8
_
2
A
P M U P U P U P U P U
3
4
4
5
C
A
0
6
_
1
A
Want to do 96-well agarose gel isolations?
Request Application Note #AN096.
Recommended
automated system
for reactions prepared
in the presence of
PCR enhancers like
betaine, DMSO, etc.
Just like the SV
system, SV 96 works
with a wide variety
of PCR additives.
I
f

i
t

w
o
r
k
s

i
n

S
V
,
i
t
l
l

w
o
r
k

i
n

S
V

9
6
.
M
a
n
u
a
l
o
r

a
u
t
o
m
a
t
e
d

9
6
-
w
e
ll
P
C
R
p
u
r
if
ic
a
t
io
n
.
29
PCR Clean-Up
Wizard
MagneSil
PCR Clean-Up System
The Wizard

(a)
removes impurities from PCR reactions, giving high
quality and yield of double-stranded amplicons. The
system removes excess nucleotides, primers and small
amplification products such as primer-dimers from PCR
reactions. The system is designed for automation on
laboratory robotic systems for unattended 96- to 384-
well purification.
PCR clean-up is performed using Promegas unique
MagneSil Paramagnetic Particles
(a)
and the MagnaBot
96 Magnetic Separation Device (Cat.# V8151) fitted with a

3/16" MagnaBot
Spacer (Cat.# V8381). The MagnaBot
96 Magnetic Separation Device is designed to work with

most robotic platforms. A MagnaBot
384 Magnetic
Separation Device is also available (Cat.# V8241).
The MagneSil PCR Clean-Up procedure is fast and
reliable. PCR products bind to MagneSil particles in
the presence of guanidine hydrochloride and remain
tightly bound during washing. Purified DNA is eluted in
water and may be used for automated fluorescent
sequencing and microarray spotting. The MagneSil
PCR Clean-Up System is ideally suited for reactions
prepared using PCR Master Mix and GoTaq DNA
Polymerase. It also works well with reactions prepared
using AmpliTaq
and AmpliTaq Gold
DNA Polymerase.
Array images after hybridization with Cy
3 and Cy
5 fluorescent control
E. coli targets. E. coli genomic DNA was amplified using 96 unique primer pairs. The
Wizard
MagneSil PCR Clean-Up System-purified PCR products were printed onto a

poly-L-lysine-coated slide and hybridized to Cy
3- or Cy
5-labeled E. coli cDNA. For

further details, see Splinter BonDurant, S. et al. (2002) MagneSil Paramagnetic Particles:
A high-throughput PCR purification system for microarrays. Promega Notes 80, 1416.
Percent recovery of PCR products purified using the Wizard
MagneSil PCR
Clean-Up System. PCR amplimers were purified from standard amplification reactions
performed using either PCR Master Mix or AmpliTaq
DNA Polymerase.
Wizard

Cat.#: A1930 (4 96 preps)
A1931 (8 96 preps)
A1935 (100 96 preps)
Protocols:
Automated 96-well plate protocol
Automated 384-well plate protocol
www.promega.com/tbs/ep009/ep009.html
For information on automated methods visit:
%

R
e
c
o
v
e
r
y
PCR Product Size (bp)
3
2
8
9
M
A
0
3
_
1
A
0
20
40
60
80
100
0 200 400 600 800 1,000
PCR Master Mix
AmpliTaq
DNA
Polymerase
3
5
7
8
T
A
1
1
_
1
A
E. coli Control Array
Cy
3 Cy
5
Quality tested for
success in fluorescent
BigDye
Sequencing
with >98% accuracy
over 600 bases read.
30
PCR Clean-Up
Wizard
PCR Preps
DNA Purification System
The Wizard

(l)
provides a simple, reliable way to purify double-stranded,
PCR-amplified DNA. PCR products are effectively
separated from contaminants, including primer-dimers
and amplification primers. This system can also be used
to purify DNA fragments from agarose gels. The DNA
can be eluted from the Wizard
PCR Preps DNA

Purification Resin in water or TE buffer, with little salt or
macromolecular contamination. A unique feature of this
resin-based method is its size cutoff capability. The resin
does not appreciably bind double-stranded DNA smaller
than 200bp, virtually assuring the removal of primer-
dimers from the reaction. Multiple PCR preps can be
easily processed at one time using the Vac-Man
Laboratory Vacuum Manifold (Cat.# A7231).

Recovery of PCR products using the Wizard
PCR Preps DNA Purification

System. Equivalent amounts of a PCR reaction taken before and after purification were
separated on a 1% agarose gel and stained with ethidium bromide.
Purification and analysis of PCR products from low-melting agarose using the
Wizard
PCR Preps System. Reactions are shown before (lanes 2 and 4) and after
(lanes 3 and 5) purification.
1,000
bp
M M
500
200
50
Before Purification After Purification
0
9
3
5
T
A
1
1
_
4
A
1 2 3 4 5
0
3
4
8
T
A
0
8
_
3
A
Wizard

Cat.#: A7170 (50 preps)
A2180 (250 preps)
Protocol:
Citations for use available at:
Centrifuge to elute
DNA.
Add resin/DNA
mix to syringe barrel
and apply vacuum.
Resin/DNA mix
Add isopropanol,
removing solution
by vacuum.
Transfer
Minicolumn to
microcentrifuge tube.
Centrifuge.
Transfer Minicolumn
to new tube.
Add water.
2
8
1
9
M
B
0
4
_
1
A
Overview of PCR product purification using the Wizard
PCR Preps DNA

Purification System and the Vac-Man
Laboratory Vacuum Manifold.

S
yring
e
-
base
d

protoc
ol
also
prov
id
e
d

(w
h
e
re

no
v
ac
uum

is
re
quire
d
).
31
PCR Clean-Up
PCR Clean-Up Systems and Accessories
Product Size Cat.#
Wizard

(k)
50 preps A9281
250 preps A9282
For Laboratory Use. Manual spin-basket system for direct PCR purification and gel isolation from standard agarose gels.
Product Size Cat.#
Wizard
SV 96 PCR Clean-Up System* 1 96 preps A9340

4 96 preps A9341
8 96 preps A9342
Vac-Man
96 Vacuum Manifold 1 each A2291

* For Laboratory Use. The Wizard
SV 96 PCR Clean-Up System provides a manual or automated system for 96-well direct PCR purification by vacuum. Compatible with a wide variety of PCR
additives and all Promega amplification reaction buffers.
Product Size Cat.#
Wizard

(a)
* 4 96 preps A1930
8 96 preps A1931
Wizard
MagneSil PCR Clean-Up System, HTP1

(a)
* 100 96 preps A1935
MagnaBot

MagnaBot

MagnaBot
Spacer (3/16) 1 each V8381

384-Well Plate, Flat 10 pack V5291
MagneSil PCR Clean-Up System provides an automated 96- or 384-well system for direct purification of PCR products. Compatible with standard reactions
using Promega's PCR Master Mix or GoTaq DNA Polymerase.
Product Size Cat.#
Wizard

(l)
* 50 preps A7170
250 preps A2180
Vac-Man
Laboratory Vacuum Manifold, 20-sample capacity 1 each A7231

PCR Preps DNA Purification System is a manual, resin-based vacuum system for direct purification or isolation from agarose gels.
Related Products
Product Size Cat.#
Agarose, LE, Analytical Grade 100g V3121
500g V3125
Agarose, Low Melting Point, Analytical Grade 25g V2111
Blue/Orange Loading Dye, 6X* 3ml (3 1ml) G1881
TAE Buffer, 10X 1,000ml V4271
TAE Buffer, 40X 1,000ml V4281
TBE Buffer, 10X 1,000ml V4251
32
Cloning PCR DNA
Overview
Cloning PCR products into plasmid vectors is a
common downstream application of PCR. When PCR
was in its infancy, researchers found that it was not easy
to clone PCR products by simple blunt-end ligation into
blunt-ended plasmid vectors because some
thermostable DNA polymerases, including Taq DNA
Polymerase, add a single nucleotide base extension to
the 3-end of blunt DNA in a template-independent
fashion (1,2). Most commonly the base added is
adenine, leaving what is called an A overhang. To
overcome this, researchers had to treat PCR products to
blunt-end the PCR fragments prior to cloning. Such
experiments often suffered from low cloning efficiencies.
Another commonly used strategy for PCR cloning is to
add restriction enzyme recognition sites to the ends of
PCR primers (3). The PCR product is then digested and
cloned into the desired vector. When using this method,
care must be exercised in primer design because not all
enzymes cleave efficiently at the ends of DNA fragments,
and you may not be able to use every enzyme you
desire (4,5). Some enzymes require extra bases outside
the restriction enzyme recognition site, adding further
expense to the PCR primers as well as increasing the
risk of annealing to unrelated sequences in the genome.
The fact that amplicons generated with Taq DNA
Polymerase typically have A overhangs led to the
method referred to as T-vector cloning. In essence, the
plasmid cloning vector is engineered to contain 3-T
overhangs that match the 3-A overhang of the amplicon
(6). The A-tailed amplicon is directly ligated to the T-
tailed plasmid vector, and there is no need for further
enzymatic treatment of the amplicon other than the
action of T4 DNA Ligase. Promega has systems based
on this technology for routine subcloning, direct
mammalian expression and bacterial expression.
References
1. Clark, J.M. (1988) Novel non-template nucleotide addition reactions
catalyzed by procaryotic and eucaryotic DNA polymerases. Nucl. Acids Res.
16, 967786.
2. Mole, S.E., Iggo, R.D. and Lane, D.P. (1989) Using the polymerase chain
reaction to modify expression plasmids for epitope mapping. Nucl. Acids
Res. 17, 3319.
3. Scharf, S.J., Horn, G.T. and Erlich, H.A. (1986) Direct cloning and sequence
analysis of enzymatically amplified genomic sequences. Science 233,
107678.
4. Kaufman, D.L. and Evans, G.A. (1990) Restriction endonuclease cleavage at
the termini of PCR products. BioTechniques 9, 3046.
5. Digestion of restriction sites close to the end of linear DNA. In: Restriction
Enzyme Resource Guide. Promega Corporation.
www.promega.com/guides/re_guide/chaptwo/2_6.htm
6. Mezei, L.M. and Storts, D.R. (1994) Cloning PCR Products. In: PCR
Technology Current Innovations. Griffin, H.G. and Griffin, A.M. (eds). CRC
Press, pp. 217.
pGEM
-T &
pGEM
-T Easy
Vectors
pTARGET Mammalian
Expression Vector
PinPoint Xa-1
T-Vector
T
A
A
T
T
T
T
T
For cloning PCR products, the choice is yours
3
7
7
9
M
A
0
7
_
2
A
Basic
Subcloning
Direct Mammalian
Expression
Direct Bacterial
Expression
33
Cloning PCR DNA
Basic Subcloning
pGEM
-T and pGEM
-T Easy Vector Systems

The most basic need in PCR cloning is for simple, general
cloning vectors. The pGEM
-T and pGEM
-T Easy Vector
Systems
(h,i)
were designed for just that purpose. The
vectors are based on the pGEM
-5Zf(+) Vector
(h)
backbone.
The pGEM
-T and -T Easy Vectors provide convenient T7

and SP6 promoters that serve as sequencing primer
binding sites and also allow in vitro transcription of either
strand of the insert with the appropriate RNA polymerase.
The vectors also have the lacZ coding region, allowing
easy blue/white screening of recombinants. The pGEM
-T
and -T Easy Vectors are provided with 2X Rapid Ligation
Buffer, which allows efficient ligation in just 1 hour with the
supplied T4 DNA Ligase. You can either supply your own
favorite E. coli competent cells or purchase the system with
Promegas high-efficiency JM109 Competent Cells. The
choice is yours.
pGEM
-T Vector System I
(you supply the competent cells)
Cat.#: A3600 (20 reactions)
pGEM
-T Vector System II
(supplied with High Efficiency JM109 Competent Cells)
Protocol:
Citations for use of the pGEM
-T System online at:

Need sequencing-grade plasmid DNA?
Promega has the system.
Wizard
Plus SV Minipreps DNA Purification System

(r,s)
Simple spin preps for plasmid DNA. Guaranteed for
fluorescent sequencing.
Cat.#: A1330 (50 preps)
Cat.#: A1460 (250 preps)
Protocol:
What is Blue/White Selection?
The enzyme -galactosidase, the product of the
bacterial lacZ gene, can be separated into two
domainsthe -fragment and the -fragment. The two
fragments interact to form a functional -galactosidase.
For blue/white selection, the -fragment is expressed
by the E. coli host strain, and the -fragment is
provided by the cloning vector. An intact, in-frame
-fragment will interact with the host strain
-fragment, creating functional -galactosidase. This is
known as -complementation. Bacteria capable of
producing functional -galactosidase will cleave the
substrate X-Gal (5-bromo-4-chloro-3-indolyl--D-
galactopyranoside), creating blue colonies when grown
on indicator plates containing IPTG and X-Gal (see
recipe on p. 39). Blue/white-capable cloning vectors
have a multiple cloning site within the -fragment
coding sequence. When your sequence of interest is
inserted within this region, the -fragment is disrupted,
-complementation does not occur, and the colony is
white. E. coli (e.g., JM109, DH5 or XL1-Blue)
transformed with an insert-containing plasmid produce
white colonies, while those containing empty or
religated vector produce blue colonies.
Sca I
1875
ori
Amp
r
Apa I
Aat II
Sph I
BstZ I
Nco I
Sac II
Spe I
Not I
BstZ I
Pst I
Sal I
Nde I
Sac I
Bst X I
Nsi I
T7
Xmn I 1994
Nae I
2692
lacZ
f1 ori
1 start
14
20
26
31
37
46
55
62
62
73
75
82
94
103
112
126
SP6
T T
pGEM
-T
Vector
(3000bp)
0
3
5
6
V
A
0
4
_
3
A
Select recombinants by
blue/white selection.
Drop out your
insert with a
single Bst Z I
digest.
S
e
q
u
e
n
c
e

in
s
e
r
t
s

w
it
h
:
S
P
6

P
r
o
m
o
t
e
r

P
r
im
e
r
T
7

P
r
o
m
o
t
e
r

P
r
im
e
r
p
U
C
/
M
1
3

F
o
r
w
a
r
d

P
r
im
e
r
p
U
C
/
M
1
3

R
e
v
e
r
s
e

P
r
im
e
r
34
Cloning PCR DNA
For maximum subcloning efficiency, purify the PCR
product before subcloning. The presence of PCR
primers and primer-dimers can reduce subcloning
efficiency. See Chapter 3 for more information.
If you do not purify your PCR product, at least make
the amplification as specific as possible. The cleaner
the product, the better the ligation efficiency. Try to
avoid production of primer-dimers by optimizing the
amplification reaction conditions. See Chapter 2 for
more information on optimizing PCR.
Example of Transformation Efficiency Calculation:
After 100l competent cells are transformed with
0.1ng uncut plasmid DNA, the transformation
reaction is added to 900l of SOC medium
(0.1ng DNA/ml). A 1:10 dilution with SOC medium
(0.01ng DNA/ml) is made, and 100l is plated on
each of two plates (0.001ng DNA/100l). If 200
colonies are obtained (average of two plates), what is
the transformation efficiency?
200cfu
1ng
= 2 10
8
cfu/g DNA
0.001ng 10
3
g
pGEM
-T Easy Vector System I

pGEM
-T Easy Vector System II

(supplied with High Efficiency JM109 Competent Cells)
Protocol:
Citations for use of the the pGEM
-T Easy System
online at:
Relationship between incubation time and cloning efficiency using the 2X
Rapid Ligation Buffer and the pGEM
-T Easy Vector. The Control Insert DNA

supplied with the pGEM
-T Easy Vector was ligated into the vector using a 1:1 vector:insert
molar ratio. The Rapid Ligation Buffer and T4 DNA Ligase were used in ligation reactions,
which were set up at room temperature (24C) and allowed to proceed from 0.25 to 16
hours. Number of white colonies (transformants) versus time of ligation are shown. This
graph was adapted from Table 2 in Frackman, S. and Kephart, D. (1999) Rapid Ligation for
the pGEM
-T and pGEM
-T Easy Vector Systems. Promega Notes 71, 810.

Sca I 1890
ori
pGEM
-T Easy
Vector
(3015bp)
Apa I
Aat II
Sph I
BstZ I
Nco I
BstZ I
Not I
Sac II
EcoR I
Spe I
EcoR I
Not I
BstZ I
Pst I
Sal I
Nde I
Sac I
Bst X I
Nsi I
T7
Xmn I 2009
Nae I 2707
lacZ
f1 ori
1
start
14
20
26
31
37
43
43
49
64
70
77
77
88
90
97
109
118
127
141
SP6
T T
1
4
7
3
V
A
0
5
_
6
A
Drop out your
insert with a
single Bst Z I,
EcoR I or
Not I digest.
Sequence inserts with:
SP6 Promoter Primer
T7 Promoter Primer
pUC/M13 Forward Primer
pUC/M13 Reverse Primer
For maximum efficiency,
use competent cells
capable of at least
1 x 10
8
cfu/g DNA.
4
0
1
9
M
A
0
3
_
3
A 0
50
100
150
200
250
300
350
400
450
0 4 8 10 12 14 16
N
u
m
b
e
r

o
f

W
h
i
t
e

C
o
l
o
n
i
e
s
Hours
2 6
35
Cloning PCR DNA
The pTARGET Mammalian Expression Vector
(i,j)
is
designed to streamline your experiments, allowing you
to go from PCR and T-vector cloning directly to
expression analysis in a mammalian system. The
pTARGET Vector is based on the popular pCI-neo
Vector
(h,j)
(Cat.# E1841) and delivers powerful
mammalian expression through the CMV promoter. The
vector also has the neomycin resistance necessary for
G-418 Sulfate selection of stable transformants.
The pTARGET Vector is the only mammalian
expression T-vector offering blue/white selection of
recombinants. The vector contains the lacZ -peptide
fragment to complement the -fragment of lacZ that is
expressed in common E. coli strains like JM109,
DH5 and XL-1 Blue. See page 33 for more
explanation of blue/white selection.
Cat.#: A1410 (20 reactions and 20 transformations
with high-efficiency JM109 Competent Cells)
Protocol:
Citations for use of the pTARGET System online at:
The pTARGET Vector contains the simian virus 40
(SV40) enhancer and early promoter region
upstream of the neomycin phosphotransferase gene.
The SV40 early promoter contains the SV40 origin of
replication, which will induce transient, episomal
replication of the pTARGET Vector in cells expressing
the SV40 large T antigen such as COS-1 or COS-7
cells (1). Consequently, the copy number of the
vector will increase in these SV40-transformed cell
lines and give higher transient expression than in
other cell types.
1. Gluzman, Y. (1981) SV40-transformed simian cells support the
replication of early SV40 mutants. Cell 23, 17582.
pTARGET Mammalian Expression Vector has been
used for transient expression in many cell lines
including:
COS-1 SV40-transformed monkey kidney
COS-7 SV40-transformed monkey kidney
H9c2 rat myoblast
Primary human melanoma
The pTARGET Mammalian Expression Vector has
been used to create stable transfectants by G-418
Sulfate selection in many cell lines including:
1376 TCC human bladder transitional cell carcinoma
293 human embryonic kidney cell
A549 human adenocarcinoma
CHO Chinese hamster ovary
NIH/3T3 mouse fibroblast
J82 human bladder transitional cell carcinoma
PS120 Chinese hamster lung fibroblast
RAW264.7 mouse monocyte/macrophage cell line
T24 human bladder transitional cell carcinoma
U937 human leukemic cells
See Promegas online citation database for further
examples and details: www.promega.com/citations/
Sgf I 664
I-Ppo I
851
Bgl II 5665
SV40 Enhancer/
EarlyPromoter
SV40 Late
poly (A)
fl ori Synthetic
poly(A)
Amp
r
ori
CMV
Enhancer/Promoter
Intron
Neo
pTARGET
Vector
(5670bp)
T
T
EcoR I
BamH I
Nhe I
Xho I
Mlu I
lacZ
lacZ
Sma I
Kpn I
Sal I
Acc I
Not I
EcoR I
T7
1250
1256
1264
1270
1276
1293
1301
1303
1304
1311
1318
T overhangs
1
5
0
5
V
A
0
7
_
6
A
S
equence inserts w
ith:
T
7
Prom
oter Prim
er
pT
ARG
ET

S
equencing
Prim
er
T
h
e

only

m
am
m
alian
e
xpre
ssion
T
-
v
e
c
tor
c
apable

of

blue
/w
h
ite
sc
re
e
nin
g
.
Drop out your insert
with a single
Eco R I digest.
36
Cloning PCR DNA
Direct Mammalian Expression (continued)
Expression of various reporter proteins using either the pTARGET Mammalian Expression Vector or the pCI-neo Mammalian Expression Vector. The pTARGET Vector
was designed from the pCI-neo Vector (Cat.# E1841). Vector sequences for blue/white selection do not interfere with expression. T.U. = Turner light units. Details of this experiment may be
found in Brondyk, B. (1996) pTARGET Vector: A new mammalian expression T-Vector. Promega Notes 58, 27.
Need transfection grade plasmid DNA?
Promega has the system.
Wizard MagneSil Tfx System
For automated 96-well transfection-grade plasmid DNA
purification.
Cat.#: A2380 (4 96 preps)
A2381 (8 96 preps)
Protocol:
For information on automated methods visit:
For expression in mammalian systems, your amplicon
should contain an initiation AUG codon and a stop
codon. Ideally the AUG codon is in the context of a
Kozak consensus sequence: (A or G)CCAUGG (1). Be
sure the initiation codon is the first AUG codon
encountered in the sequence.
1. Kozak, M. (1987) At least six nucleotides preceding the AUG initiator
codon enhance translation in mammalian cells. J. Mol. Biol. 196, 94750.
20
40
60
80
100
120
140
0
H
u
m
a
n

G
r
o
w
t
h

H
o
r
m
o
n
e

A
c
t
i
v
i
t
y

(
n
g
/
m
l

c
o
n
d
.

m
e
d
i
u
m
)
F
i
r
e
f
l
y

L
u
c
i
f
e
r
a
s
e

A
c
t
i
v
i
t
y

(
T
.
U
.
/
p
l
a
t
e

1
0
6
)
Cells Transfected
C
A
T

A
c
t
i
v
i
t
y

(
C
P
M
/
p
l
a
t
e

1
0
6
)
-
g
a
l
a
c
t
o
s
i
d
a
s
e

A
c
t
i
v
i
t
y

(
u
n
i
t
s
/
p
l
a
t
e

1
0
-
3
)
1
2
3
4
5
6
7
8
0
pCI-neo
pTARGET
pCI-neo
pTARGET
pCI-neo
pTARGET
pCI-neo
pTARGET
pCI-neo
pTARGET
R
e
n
i
l
l
a

L
u
c
i
f
e
r
a
s
e

A
c
t
i
v
i
t
y

(
T
.
U
.
/
p
l
a
t
e

1
0
6
)
HeLa NIH3T3
Cells Transfected
HeLa NIH3T3
Cells Transfected
HeLa NIH3T3
Cells Transfected
HeLa NIH3T3
Cells Transfected
HeLa NIH3T3
2.5
2
3.5
3
1.5
0.5
1
0
10
20
30
40
50
60
0
0
5
10
15
20
25
30
35
40
A. C. B.
D. E.
1
5
3
9
M
A
0
7
_
6
A
Learn about transfection and tools available from
Promega in the Transfection Guide available online at:
www.promega.com/guides/, or request literature
#BR041 from your local Promega representative.
Experimental details
available at:
www.promega.com/pnotes/
58/5189a/5189a.html
37
Cloning PCR DNA
PinPoint Xa-1 T-Vector Systems
The PinPoint Xa-1 T-Vector
(h,i,t)
provides a convenient
means to clone and express PCR products in E. coli.
Fusion proteins made using the PinPoint Xa-1
T-Vector are easily expressed and purified. The
PinPoint Xa-1 T-Vector carries a segment encoding
a polypeptide that becomes biotinylated in E. coli and
subsequently functions as a purification tag (1).
Biotinylated fusion proteins produced with this system
can be affinity-purified using the SoftLink Soft Release
Avidin Resin
(u,v)
(Cat.# V2011). This proprietary resin
allows elution of the fusion protein under nondenaturing
conditions. The TetraLink Tetrameric Avidin Resin
(w)
(Cat.# V2591) also can be used to capture biotinylated
proteins irreversibly as a way to generate affinity resins.
Reference
1. Cronan, J.E., Jr. (1990) Biotination of proteins in vivo. A post-translational
modification to label, purify, and study proteins. J. Biol. Chem. 265,
1032733.
Protein expression using the PinPoint Xa-1 T-Vector System. Use with
SoftLink Soft Release Avidin Resin (Cat.# V2011) to purify fusion proteins. For details,
see the PinPoint Xa-1 T-Vector System Technical Bulletin #TB234.
PinPoint Xa-1 T-Vector System I
Cat.#: V2610 (20 reactions)
Pinpoint Xa-1 T-Vector System II
(supplied with high-efficiency JM109 Competent Cells)
Cat.#: V2850 (20 reactions and 20 transformations
with JM109 Competent Cells)
Protocol:
Citations for use of the PinPoint Xa-1 T-Vector
System online at:
Note: Amplification primers must be designed to clone
in frame with the PinPoint Fusion Protein. Full
details are provided in Technical Bulletin #TB234.
389
392
400
404
410
414
422
430
435
452
Sal I 52
BstX I 264
Nde I
532
Factor
Xa site
379-390
Xmn I 1106
Sca I 1225
Pvu I 1337
Fsp I 1483
ori
Sac I
377
Hpa I 2777
T7 promoter Cla I 3018
tac promoter
EcoR I 3277
Pst I 3296
SP6
Nru I
Hind III
Pvu II
BamH I
Acc65 I
Kpn I
Bgl II
Sma I
Not I
purification
tag sequence
1-386
Amp
r
PinPoint

Xa-1
T-Vector
(3331bp)
1
1
1
8
V
A
0
7
_
5
A
1. Wash.
2. Elute with free biotin.
PinPoint
Expression Vector
1. Express fusion protein in E. coli.
2. Lyse cells, digest with nuclease.
3. Pellet cell debris.
Biotinylated
fusion protein
in crude cell
supernatant
Detection using
avidin reagents
Proteolytic removal of
biotinylated tag peptide
0
3
6
7
M
C
0
3
_
3
A
Bind to SoftLink Soft Release
Avidin Resin.
Sequence inserts with:
SP6 Promoter Primer
PinPoint Vector
Sequencing Primer
38
Cloning PCR DNA
PCR Cloning Techniques
Rapid PCR-Based Screen for Orientation of Insert
All Promega PCR cloning vectors have some unique
landmarks, including RNA promoter primer binding sites
allowing easy sequencing of inserts. The primer binding
sites can also be used to rapidly screen for insert
orientation. For example, we cloned a 1.8kb insert into
the pGEM
-T Easy Vector System (1). The insert could

be oriented in one of two waystoward the T7
promoter or toward the SP6 promoter:
To check for orientation, we performed colony PCR with
the T7 Promoter Primer and either the gene-specific
forward PCR primer or reverse PCR primer. Eight white
colonies with inserts were chosen from the cloning
experiment for orientation analysis. Clones with the T7
orientation will produce the fragment with only the
reverse PCR primer, and clones in the opposite (SP6)
orientation will only produce a product with the forward
PCR primer as illustrated below.
Reference
1. Knoche, K. and Kephart, D. (1999) Cloning blunt-end Pfu DNA polymerase-
generated PCR fragments into pGEM
-T Vector Systems. Promega Notes

71, 1013.
Giving Blunt-Ended DNA an A-Tail for T-Vector
Subcloning
PCR amplicons generated with proofreading
polymerases like Pfu or Tli DNA Polymerase are blunt-
ended. Promega has developed an easy method to add
an A-tail to PCR products generated using these
polymerases so that they will become suitable
substrates for T-vector cloning. Full details of the
protocol are available in the pGEM
-T and pGEM
-T
Easy Vector Systems Technical Manual #TM042.
Ends Left by Various Thermostable Polymerases.
Taq DNA Polymerase 3A overhang*
GoTaq DNA Polymerase 3A overhang*
Tfl DNA Polymerase 3A overhang*
Tth DNA Polymerase 3A overhang*
Pfu DNA Polymerase Blunt
Tli DNA Polymerase Blunt
Long PCR mixes Blunt
Proofreading Polymerases Blunt
* All bases may be found at 3 overhang. A tends to occur most often.
T7 Orientation
Forward Primer
Reverse Primer SP6 Primer
T7 Primer
T7 1.8kb fragment SP6
SP6 Orientation
Reverse Primer
Forward Primer SP6 Primer
T7 Primer
T7 1.8kb fragment SP6
2
5
7
4
M
B
0
2
_
9
A
Start with 17l of purified PCR fragment generated by a
proofreading polymerase (e.g., Pfu DNA Polymerase).
Add 1l Taq DNA Polymerase 10X Reaction Buffer with MgCl
2
.
Add dATP to a final concentration of 0.2mM.
Add 5 units of Taq DNA Polymerase.
Add deionized water to a final reaction volume of 10l.
Incubate at 70C for 1530 minutes.
Use 12l in a ligation reaction with
Promegas pGEM
-T and pGEM
-T Easy Vector.
2
3
5
7
M
A
0
2
_
9
A
An A-tailing procedure for blunt-ended PCR fragments.
Colony PCR. Colonies were suspended in 50l sterile water, boiled for 10 minutes,
centrifuged at 16,000 g for 5 minutes, and 5l of the supernatant was used in each
amplification. The DNA was amplified by PCR in 50l volumes with 50pmol of each primer
and 1.25 units of Promegas Taq DNA Polymerase (Cat.# M1661). After an initial
denaturation of 2 minutes at 94C, the amplification profile was 35 cycles of denaturation
(94C for 30 seconds), annealing (55C for 1 minute) and extension (72C for 2.5
minutes); PCR was concluded with 1 cycle of 72C for 10 minutes. Amplification products
(8l) were analyzed on a 1% agarose gel containing ethidium bromide.
M 1 2 3 4
2
5
7
5
T
A
0
2
_
9
A
T7 + For.
bp
2,645
1,605
1,198
676
517
222
T7 + Rev.
1 2 3 4
For more information and techniques
for cloning PCR DNA, check out
Promega's Frequently Asked Questions
on the T-vector cloning systems at:
www.promega.com/faq/
39
Cloning PCR DNA
PCRCloning Techniques (continued)
What PCR Cloning Controls Can Do For You
Each Promega PCR cloning system is provided with a
Control Insert. The ligation and subsequent transformation
of the Control Insert can give you a lot of information
about your ligation and transformation reactions.
The total number of blue colonies in Control Insert and
no-insert controls should be approximately equal. The
negative control may have some white colonies.
Bacterial Plates for Blue/White Selection
LB medium (per liter)
10g Bacto
-tryptone
5g Bacto
-yeast extract
5g NaCl
Adjust pH to 7.0 with NaOH.
Ampicillin Stock Solution
Dissolve at 50mg/ml in water. Filter sterilize. Store in
aliquots at 20C
IPTG stock solution (0.1M)
1.2g IPTG (Cat.# V3951)
Add water to 50ml final volume. Filter-sterilize and
store at 4C.
X-Gal (2ml)
100mg X-Gal (Cat.# V3941)
Dissolve in 2ml N,N-dimethyl-formamide. Cover with
aluminum foil and store at 20C.
LB plates with ampicillin/IPTG/X-Gal
Add 15g agar to 1 liter of LB medium. Autoclave.
Allow the medium to cool to 50C before adding
ampicillin to a final concentration of 100g/ml, then
supplement with 0.5mM IPTG and 80g/ml X-Gal and
pour the plates. Pour 3035ml of medium into 85mm
petri dishes. Let the agar harden. Store at 4C for up
to 1 month or at room temperature for up to 1 week.
Interpreting Results
Results with the experimental insert look like those
with the Control Insert in terms of efficiency and %
white colonies.
Successful experiment. Greater than 80% of the white
colonies should contain inserts.
Results with the experimental insert and Control
Insert look like the negative control.
Ligation has failed. Avoid multiple freeze/thaws of the
ligation buffer. Ligase buffer contains ATP and could
be damaged by freeze-thaws. You may need to aliquot
the ligase buffer into useful portions for your
experimental needs.
Few/No colonies with experimental insert, Control
Insert or negative control.
Transformation has failed. Reassess the competent
cells with an intact, supercoiled plasmid and
determine the transformation efficiency. Use cells
>1 10
8
cfu/g to insure >100 colonies from the
Control Insert ligation.
Experimental insert gives more blue colonies than
the Control Insert or negative control and less white
colonies than the Control Insert.
In-frame insertion with no interruption of the
-fragment. Although the pGEM
-T Vector Control
Insert will produce recombinants that generate white
colonies, the insertion of other DNA fragments into the
lacZ coding sequence may not result in white colonies
unless the fragments disrupt the lacZ reading frame.
Although this tends to occur most frequently with PCR
products of 500bp or less, inserts of up to 2kb have
been reported to result in blue colonies. Moreover,
some insert DNAs can also give pale blue colonies or
bulls eye colonies that have a blue center and a
white perimeter. In one case, we found that a 1.8kb
insert produced white colonies when oriented in one
direction and bulls eye colonies when oriented in the
opposite direction (1).
1. Knoche, K. and Kephart, D. (1999) Cloning blunt-end Pfu DNA
polymerase-generated PCR fragments into pGEM
-T Vector Systems.
Typical PCR Cloning Results Using pGEM
-T
Easy Vector and JM109 Competent Cells
(1.5 10
8
cfu/ng DNA).
Efficiency % White
(cfu/ng DNA) Colonies
Control Insert 1,110 92%
Control Insert 1,125 92%
No insert 92 0%
No insert 109 0%
Ligations performed at room temperature for 1 hour.
40
Cloning PCR DNA
Basic Subcloning
Product Size Cat.#
pGEM
-T Vector System I
(h,i)
20 reactions A3600
pGEM
-T Vector System II
(h,i)
20 reactions A3610
pGEM
-T Easy Vector System I

(h,i)
20 reactions A1360
pGEM
-T Easy Vector System II

(h,i)
20 reactions A1380
For Laboratory Use. pGEM
-T and pGEM
-T Easy Vector Systems I do not include competent cells. With System II, competent cells are provided.
Product Size Cat.#
(i,j)
20 reactions A1410
Competent Cells provided.
Product Size Cat.#
PinPoint Xa-1 T-Vector System I
(h,i,t)
(you provide competent cells) 20 reactions V2610
PinPoint Xa-1 T-Vector System II
(h,i,t)
(includes competent cells) 20 reactions V2850
SoftLink Soft Release Avidin Resin
(u,v)
1ml V2011
5ml V2012
TetraLink Tetrameric Avidin Resin
(w)
1ml V2591
5ml V2592
Primers
Product Size Cat.#
T7 Promoter Primer (10g/ml)[5-d(TAATACGACTCACTATAGGG)-3] 2g Q5021
SP6 Promoter Primer (10g/ml) [5-d(TATTTAGGTGACACTATAG)-3] 2g Q5011
pUC/M13 Forward Primer (10g/ml) [5-d(CGCCAGGGTTTTCCCAGTCACGAC)-3] 2g Q5601
pUC/M13 Reverse Primer (10g/ml) [5-d(TCACACAGGAAACAGCTATGAC)-3] 2g Q5421
pTARGET Sequencing Primer (10g/ml) [5-d(TTACGCCAAGTTATTTAGGTGACA)-3] 2g Q4461
PinPoint Vector Sequencing Primer [5-d(CGTGACGCGGTGCAGGGCG)-3] 2g V4211
DNA Purification Systems
Product Size Cat.#
Wizard
Plus SV Minipreps DNA Purification System

(r,s)
* 50 preps A1330
250 preps A1460
Wizard MagneSil Tfx System 4 96 preps A2380
(Automated transfection-grade plasmid purification.) 8 96 preps A2381
41
Cloning PCR DNA
Accessory Products
Product Size Cat.#
Select96 Competent Cells (Single aliquot competent cells, use 1 to 96 at a time, >1 10
8
cfu/g DNA) 1 96 preps L3300
JM109 High Efficiency Competent Cells (10
8
cfu/g)* (Packaged 5 200l; use 50l per transformation >1 10
8
cfu/g DNA) 1ml L2001
IPTG, Dioxane-Free 5g V3951
50g V3953
X-Gal (50mg/ml) 100mg V3941
Antibiotic G-418 Sulfate (potency >500g/mg) 100mg V7981
1g V7982
5g V7983
Antibiotic G-418 Sulfate Solution (potency 4060mg/ml) 20ml V8091
Transfection Reagents
Product Size Cat.#
TransFast Transfection Reagent
(x)
1.2mg E2431
Tfx-10 Reagent
(y)
9.3mg E2381
Tfx-20 Reagent
(y)
4.8mg E2391
Tfx-50 Reagent
(y)
2.1mg E1811
42
(a)
U.S. Pat. Nos. 6,027,945 and 6,368,800, Australian Pat. No. 732756 and other patents and patents pending.
(b)
This product may be covered by one or more of the following patents issued to Promega Corporation for multiplex amplification of STR loci: U.S. Pat. Nos. 5,843,660, 6,479,235 and
6,221,598 and Australian Pat. No. 724531. Other patents are pending.
(c)
U.S. Pat. No. 6,238,863 and other patents pending.
(d)
STR loci are the subject of U.S. Pat. No. 5,766,847, German Pat. No. DE 38 34 636 C2 and other patents issued to the Max-Planck-Gesellschaft zur Frderung der Wissenschaften, eV,
Germany. The development and use of STR loci are covered by U.S. Pat. No. 5,364,759, Australian Pat. No. 670231 and other pending patents assigned to Baylor College of Medicine,
Houston, Texas.
Use of Promega's STR Systems requires performance of the polymerase chain reaction (PCR), which is the subject of European Pat. Nos. 201,184 and 200,362 and U.S. Pat. Nos. 4,683,195,
4,965,188 and 4,683,202 owned by Hoffmann-La Roche. Purchase of Promega's STR Systems does not include or provide a license with respect to these patents or any other PCR-related
patent owned by Hoffmann-La Roche or others. Users of Promega's STR Systems may, therefore, be required to obtain a patent license, depending on the country in which the system is
used. For more specific information on obtaining a PCR license, please contact Hoffmann-La Roche.
(e)
Certain applications of this product are covered by patents issued and applicable in certain countries. Because purchase of this product does not include a license to perform any patented
application, users of this product may be required to obtain a patent license depending upon the particular application and country in which the product is used.
(f)
The PCR process is covered by patents issued and applicable in certain countries. Promega does not encourage or support the unauthorized or unlicensed use of the PCR process. Use of
this product is recommended for persons that either have a license to perform PCR or are not required to obtain a license.
(g)
U.S. Pat. No. 6,242,235 and other patents pending.
(h)
U.S. Pat. No. 4,766,072.
(i)
Licensed under one or more of U.S. Pat. Nos. 5,487,993 and 5,827,657 and European Pat. No. 0 550 693.
(j)
The CMV promoter and its use are covered under U.S. Pat. Nos. 5,168,062 and 5,385,839 owned by the University of Iowa Research Foundation, Iowa City, Iowa, and licensed FOR
RESEARCH USE ONLY. Commercial users must obtain a license to these patents directly from the University of Iowa Research Foundation.
(k)
U.S. Pat. Nos. 5,658,548 and 5,808,041, Australian Pat. No. 689815 and other patents pending.
(l)
Licensed under U.S. Pat. No. 5,075,430.
(m)
U.S. Pat. No. 5,552,302, Australian Pat. No. 646803 and other patents.
(n)
U.S. Pat. Nos. 4,966,964, 5,019,556 and 5,266,687, Australian Pat. Nos. 616881 and 641261 and other pending and issued patents, which claim vectors encoding a portion of human
placental ribonuclease inhibitor, are exclusively licensed to Promega Corporation.
(o)
U.S. Pat. Nos. 5,324,637, 5,492,817 and 5,665,563, Australian Pat. No. 660329 and other patents.
(p)
The method of recombinant expression of Coleoptera luciferase is covered by U.S. Pat. Nos. 5,583,024, 5,674,713 and 5,700,673.
(q)
The use of dUTP in combination with UNG is covered by patents issued and applicable in certain countries. Promega does not encourage or support the unauthorized or unlicensed use of
this process. Use of this product is recommended for persons that either have a license to perform this procedure or are not required to obtain a license.
(r)
U.S. Pat. No. 5,981,235 and Australian Pat. No. 729932 have been issued to Promega Corporation for methods for isolating nucleic acids using alkaline protease. Other patents are pending.
(s)
Australian Pat. No. 730718 and Singapore Pat. No. 64532 have been issued to Promega Corporation for an improved filtration system and method. Other patents are pending.
(t)
For research purposes only. Not for diagnostic or therapeutic use. For nonresearch uses of the portion of the vector encoding the biotinylation sequence, please contact Promega Corporation
for licensing information. For bulk purchases of the SoftLink Resin, contact TosoHaas, 156 Keystone Drive, Montgomeryville, PA 18936, 1-800-366-4875 or 215-283-5000.
(u)
For research purposes only. Not for diagnostic or therapeutic use. For bulk purchases of this product, contact TosoHaas, 156 Keystone Drive, Montgomeryville, PA 18936, 1-800-366-4875 or
215-283-5000.
(v)
Exclusively licensed under U.S. Pat. Nos. 5,276,062 and 5,395,856.
(w)
For research purposes only. Not for diagnostic or therapeutic use. For nonresearch uses of the TetraLink Resin, contact TosoHaas, 156 Keystone Drive, Montgomeryville, PA 18936,
1-800-366-4875 or 215-283-5000.
(x)
The cationic lipid component of the TransFast Transfection Reagent is covered by U.S. Pat. Nos. 5,824,812, 5,869,715 and 5,925,623, Australian Pat. No. 713093 and pending foreign
patents.
(y)
The cationic lipid component of the Tfx Reagents is covered by U.S. Pat. Nos. 5,527,928, 5,744,625 and 5,892,071, Australian Pat. No. 704189 and other pending foreign patents.
MagnaBot, pGEM, TNT, Vac-Man and Wizard are trademarks of Promega Corporation and are registered with the U.S. Patent and Trademark Office. GoTaq, ImProm-II, MagneSil, PinPoint,
pTARGET, Select96, SoftLink, TaqBead, TetraLink, Tfx and TransFast are trademarks of Promega Corporation. Amplification Assistant is a service mark of Promega Corporation.
ABIPRISM and Genotyper are registered trademarks of Applera Corporation. AmpliTaq and AmpliTaq Gold are registered trademarks of Roche Molecular Systems, Inc. Bacto is a registered
trademark of Difco Laboratories, Detroit, Michigan. BigDye is a registered trademark of Applera Corporation. Biomek is a registered trademark of Beckman Instruments, Inc. Cy is a registered
trademark of Amersham Biosciences Ltd. DH5 is a trademark of Life Technologies, Inc. Eppendorf is a registered trademark of Eppendorf-Netheler-Hinz GmbH. Ficoll is a registered trademark
of Amersham Biosciences Ltd. Finnpipette is a registered trademark of Labsystems Oy. MultiPROBE is a registered trademark of the Perkin Elmer Corporation. Oxford Benchmate is a registered
trademark of Sherwood Medical Company. Pipetman is a registered trademark of Rainin Instrument Co. Platinum is a registered tradeark of Invitrogen Corporation. Triton is a registered
trademark of Union Carbide Chemicals & Plastics Technology Corporation. Tween is a registered trademark of ICI Americas, Inc. Vent is a registered trademark of New England Biolabs, Inc.

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Genomic DNA Purification Kit

SV and SV 96 Genomic DNA Purification Systems

Magnetic 96 DNA Plant System

SV Gel and PCR Clean-Up System

SV 96 PCR Clean-Up System

MagneSil PCR Clean-Up System

PCR Preps DNA Purification System

Genomic DNA Purification

Genomic DNA Purification Kit.

Genomic DNA Purification Kit Technical Manual #TM050. DNA

Genomic DNA Purification Kit

Genomic DNA Purification Kit has been

SV and SV 96 Genomic DNA Purification

2000 with a suitable vacuum

SV Genomic DNA Purification System. Genomic DNA was

SV Genomic DNA Purification System Technical Bulletin #TB302. One microliter of

SV and SV 96 Genomic DNA Purification Systems. Promega Notes

SV Genomic DNA Purification Systems.

SV Genomic DNA Purification System spin and

SV Genomic DNA Purification System

SV and SV 96 Genomic DNA

SV and SV 96 Genomic DNA

SV and SV 96 Genomic DNA Purification Systems. Promega Notes 81, 913.

SV 96 Genomic System is suitable for

310 Genetic Analyzer, and analyzed using GeneScan

Magnetic 96 DNA Plant System

Magnetic 96 DNA Plant System uses MagneSil

FX and other robots are available.

Magnetic 96 DNA Plant System produces PCR-quality DNA from

Magnetic 96 DNA Plant System. Promega Notes 79, 2528.

Magnetic 96 DNA Plant System.

Magnetic 96 DNA Plant System.

Magnetic 96 DNA Plant System

Magnetic 96 DNA Plant

Genomic DNA Purification Kit

Laboratory Vacuum Manifold, 20-sample capacity 1 each A7231

Laboratory Vacuum Manifold can be used to process up to 20 samples at once. The

Laboratory Vacuum Manifold with the Wizard

SV Genomic DNA Purification System.

96 Vacuum Manifold 1 each A2291

96 Vacuum Manifold is required for use with the Wizard

SV 96 Genomic DNA Purification System.

96 Magnetic Separation Device* 1 each V3031

Spacer, 1/8 inch 1 each V8581

FX. The Deep Well MagnaBot

Magnetic 96 DNA Plant System

96 Magnetic Separation Device 1 each V8151

Spacer 1 each V8381

Magnetic DNA Plant System requires use of the MagnaBot

96 Magnetic Separation Device and the MagnaBot

Spacer for manual or automated DNA purification.

Taq Tfl Tth (Tli) Vent

X-100, have the additional benefit of being

MagneSil PCR Clean-Up System

SV 96 PCR Clean-Up System A9340 Yes Yes

SV Gel and PCR Clean-Up System

PCR Preps DNA Purification System

T7 Quick for PCR DNA

X-100 (Taq DNA Polymerase in Storage

-20 and NP-40 (Taq DNA

Promega 10 0.510l P-2; P-10 0.510l 0.510l Digital

SV Gel and PCR Clean-