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Cloning Protocol

Ian Hewson 2002 (modified from protocol in Promega P-gemT Easy Vector Cloning kit)

Preparation for Cloning:

Ampicillin
1g ampicillin
10 ml deionised water

Add amp to DIW, filter sterilize through 0.2 um syringe filter. Aliquot into 1 mL aliquots
and store at – 20 deg C until use

LB media
10g Tryptone
5g Yeast Extract
5g NaCl
1000ml deionised water

Make 2 x 500 mL volumes for every 2 samples to clone. Autoclave then cool to 50 deg.
C. Add 1 mL 100 mg /ml ampicillin to make 100 ug / ml LB / amp. To one of the 500 ml
volumes add 15 g agar , when cool add ampicillin then pour 25 ml into petri dishes (75
mm variety).
NOTE: if you want to use S-Gal instead of X-Gal then use the entire contents of one of
the commercially available S-Gal mixes, Just remember to add ampicillin when cool.
You should have ~ 4 plates per sample to be cloned.

SOC media
2g tryptone
0.5g yeast extract
1ml 1M NaCl (make first)
0.25ml 1M KCl (make first)
97mL deionised water

Dissolve then autoclave. On its own, stable indefinitely at 4 deg C. Day of use, make up
2M Glucose (50 mL) and 50mL 2M Mg solution (10.15g MgCl2-6H20 + 12.35g
MgSO4-7H20 + 25 mL DIW). Filter each of the glucose solution and Mg solution. Add
1mL of each of glucose and Mg solutions to the SOC media, then filter sterilize (0.22
um) the entire media. Store at 4 deg C until 1h before use.

IPTG
0.12 g isopropyl-b-d-thiogalactopyranoside (IPTG)
5 mL deionised water

Dissolve then filter sterilize through 0.22 um. store at 4 deg C. stable for many months
X-Gal
0.10g 5-bromo-4-chloro-3-indoly-b-d-galactoside (X-gal)
2 mL N,N-dimethylformamide

Dissolve, cover in foil and store (stable for many months; do not filter)

LAIX plates (LB/Ampicillin/IPTG/X-Gal)


Take LB / amp plates into laminar flow, spread 100 ul IPTG onto plates and let adsorb by
placing inverted in 37 deg C incubator for 30 min. After this, spread 20 ul of X-Gal onto
plates, then let adsorb again at 37 deg C. Store plates at 4 deg C. Good for 1 week.

Toothpicks
Cut toothpicks into 2 pieces, put in beaker and autoclave with alfol over the top.

Growth blocks
Cover 96 – well polypropylene growth blocks in alfoil, then autoclave

Also make sure you have ~ 100 eppendorf tubes (0.5 mL + 1.7 mL), and enough 5 mL
round bottomed falcon tubes with which to work. We also use 1.7 ml Eppendorf Tubes if
colony number < 96.

Cloning Protocol
(adapted from p-GemT Easy Vector (Promega) kit)

Preparation of Amplimer

Dilute your target DNA to 2.5 ng/ul in DIW.

Make the master mix:


10 X PCR Buffer 10 ul
MgCl2 25mM 10 ul
dNTPs (nucleotide mix) 2.5 ul
Primer 58 2 ul
Primer 20 2 ul
BSA (40 ng/ul) 1 ul
DIW 67.5 ul
Taq (Promega) 1 ul
Sample 4 ul

With unamended negative control.


Use the following cycling conditions:

No hot start (you are band-isolating later)


Denature 94 deg C for 30s
Anneal 56 deg C for 30 s
Extend 72 deg C for 90 s
with a final extension step of 72 deg C for 4 min
4 deg C chill at end of cycle

Run 25 cycles

After PCR is finished, run a deep-well gel (done by using 60 mL of 0.5 X TBE + 1.5 %
NuSieve agarose and using one of the small gel castings taped around the gaps – this stuff
is really viscous). After gel has set (may take a while since gel temp is very low), put I
gel box with 0.5 X TBE. Load the entire contents of the PCR reaction into one well + 10
ml loading dye mixed in the PCR tube. Run gel at 100 V for 1 h. Stain gel for 30 min
with 20 ul / ml SYBR Gold.

Take stained gel to large transilluminator (one with plastic shield). Prepare one 2 mL
tubes for each sample. Wearing UV-resistant face mask and gloves, turn on
transilluminator and off lights. Acting quickly (shortwave radiation causes formation of
primer-dimers), excise each band and place in a 2ml tube. These can be stored for later
use at –20 deg C, but in my experience it is better to get all the way through to ligation on
day one.

Extract excised bands using QIAGEN MinElute gel extraction kit (see kit protocol).

Quantify DNA after elution using PICO green kit (see separate protocol).

If low or no DNA, do not proceed.

Calculate how much DNA you will need downstream using the following equation:

50 ng insert x insert size in kb


3.0 kb vector x 3/1 = amt required

If ~ 10 ng/ul or more of product is obtained, proceed to next step. I have ligated using a
ratio as low as 1/8 (successfully).

Ligation

Set up one ligation reaction per sample to be cloned, plus one background, and one
positive control.
Before starting, if the Easy Cloning Vector II kit is new, aliquot the supplied ligation
buffer into 100 ul aliquots and store at –80 deg C. This is really sensitive to freeze-thaw.

Set up the following ligation reactions:

Sample Positive Background


2X rapid ligation buffer 5 ul 5 ul 5ul
pGem-T Easy Cloning Vector (50ng) 1 ul 1 ul 1 ul
PCR Product 3ul max - -
Control insert DNA - 2 ul -
T4 ligase 1 ul 1 ul 1ul
DIW - 1 ul 3 ul

Mix reactions by mixing with pipette tip – BE GENTLE. Use 0.5 mL autoclaved tubes
for ligations!

Put in refrigerator (4 deg C) overnight.

Transformation

Begin starting transformation, pre-heat 37 deg C shaker and also heat a water bath to 42
deg C EXACTLY!! You also need ice from the maker on Level 3.

1. Remove ligations from refrigerator and equilibriate to room temp (1 min).


Centrifuge if necessary to the bottom of the tube.

2. Add 2ul of each ligation to the bottom of a sterile 5 ml falcon round- bottomed
tube which has been pre-cooled on ice. Make sure tubes are labeled

3. Remove JM109 competent cells from freezer and place in a 50 % ice / 50 % DIW
bath for EXACTLY 5 minutes. Mix by flicking GENTLY. Talk nicely to cells!

4. Add 50 ul competent cells to the falcon round- bottomed tubes on ice using wide-
bore pipette tips and pipetting gently. flick gently to mix.

5. Leave on ice for 20 minutes.

6. Heatshock cells for EXACTLY 45 s at 42 deg C. Time is very very important!

7. Return to ice for 2 minutes EXACTLY

8. Add 950 ul SOC media (see first part of this protocol) to each transformation and
mix by flicking GENTLY. Close tubes completely (airtight). Put in incubator-
shaker (37 deg C) for 90 minutes.
9. After 60 minutes, put LAIX plates in 37 deg C incubator oven (i.e. not shaker)
inverted with lids off to dry out for 30 mins.

10. After the plates are dry and the transformations have incubated for 90 minutes in
the shaker, take to laminar flow hood. Using sterile pipette tips, spread 150 ul of
the transformation cultures onto the LAIX plates. I do about 4 plates per sample
(excess colonies are obtained, but plates can be kept at 4 deg C for later picking
and growing).

11. Seal plates with parafilm and place in 37 deg C oven overnight.\

Colony screening and liquid culture

12. After plates have incubated, place in 4 deg C fridge for 30 min (allowes easier
screening of blue / white colonies.

13. Prepare a growth block (96 well) by pipetting 1.2 mL of LB + Amp liquid media
into each well using the 1250 ul multipipettor . You can use a reagent reservoir
for this since you will have excess LB + Amp. Prepare as many blocks as you
have samples.

14. Using sterile toothpicks, pick up WHITE colonies (not blue or light blue) and
drop toothpick into growth block. Do this for as many colonies as you can find on
the plate.

15. When all wells are full, seal with 96 well- sealing tape , making sure all wells are
covered and sealed around the edges. Using a sterile 32 G needle, prick two small
holes on the top of each well.

16. Place growth blocks into the 37 deg C incubator – shaker and incubate overnight.

See “Cheap protocol for plasmid preps from clones” for next steps in preparing
plasmids, and “ checking for inserts” for how to check for inserts.