1

BL5106 Basic Molecular Genetics

Laboratory Report
Cho Yu Hwa, Lee Hee Jun, Leow Shu Hui, and Ong Sze Min
M14504

Introduction
A plasmid is a small, circular, double-stranded DNA molecule that is exist in cells as
extrachromosomal genomes, distinct from a cells chromosomal DNA, and has the ability to replicate
itself independently within a cell. Most commonly found in bacteria, plasmids are widely used as
vectors in molecular cloning, as tools to clone, transfer and manipulate genes. Plasmids used in
genetic engineering is the basis of recombinant DNA technology that provide great functionality in
molecular science, such as disease models and gene therapy. In this practical report, some of the
most commonly used techniques of Recombinant DNA technology will be discussed with detailed
steps and explanations, followed by an application of how those techniques can be used to sub-clone
a new strain of bacteria with gene of interest, in this case, the Green Flourescent Protein (GFP) gene
from pUCAG plasmid of E.coli.

Isolation of Plasmid (Practical 1a)

This step prepare plasmid DNA from relatively small volume of E.coli by using the alkaline lysis
method.
[Refer to appendix for materials and procedure]

Step Explanation

1 Buffer P1: Resuspension buffer.

The purpose of this buffer is to provide an optimal starting pH of 8.0 and an ideal condition
for subsequent lysis.
EDTA action results in inactivation of many enzymes which may harm plasmid DNA.
Tris.Cl acts as a buffering agent and maintain the pH of the resuspension buffer 8.0.
RNase A is a very stable enzyme and is active under very stringent condition
including high alkaline condition, presence of detergent and chelating agent (EDTA).
Addition of RNase A in resuspension buffer helps to remove RNA from the plasmid
preparation.

2 Sodium dodecyl sulphate (SDS) in Buffer P2 lyses bacteria cell membrane by solubilizing
the phospholipid, and denatures protein, leading to lysis and release of the cell contents.
Denaturing action of SDS also releases protein from DNA, leaving the DNA (both genomic
and plasmid DNA) free from proteins.
NaOH denatures the DNA. Cellular DNA becomes linearized and the strands are separated,
forming single-stranded linear DNA. Plasmid DNA is circular and remains topologically
constrained.
But, lysis reaction should not proceed for more than 5 min to prevent release of
chromosomal DNA and irreversible denaturation of plasmid, which would make it resistant
to restriction enzyme digestion.

3 Addition of buffer N3, which contains acidic potassium acetate, bring the solution pH back to
normal, resulting in precipitation of protein and genomic DNA. Both plasmid and genomic
DNA renatures upon addition of neutralization buffer. While plasmid DNA renatures in
correct conformation due to its circular and covalent nature, therefore, remains in the
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solution, genomic DNA precipitates due to random asociation of both the strands.
SDS reacts with potassium acetate and form insoluble potassium dodecyl sulfate (KDS).
This will allow for the easy removal of the SDS from your plasmid DNA.
Solution turns cloudy due to precipitation of cellular debris, denatured proteins, SDS and
linear DNA.

4 Precipitate formed upon addition of neutralization solution is separated into distinct pallet at
the bottom of the tube by centrifugation at high speed. The plasmid DNA is in the
supernatant, which will be used for the subsequent steps.

5 QIAprep spin column: Contains silica membrane that allows selective adsorption of plasmid
DNA in the presence of a high concentration of chaotropic salt, and allows elution of high-
purity DNA in a small volume of low-salt buffer.
Flow-through contains impurities such as RNA and cellular proteins since DNA would have
binded to QIAprep spin column.

6 Buffer PB: Provides optimal salt concentration of chaotropic salts (disrupt hydrogen bonding
in surrounding water molecules) and pH for adsorption of plasmid DNA to QIAprep spin
column membrane. It also removes endonucleases to ensure that DNA is not degraded.

7 Buffer PE: Contains non-chaotropic salts and large amounts of alcohol which remove any
remaining chaotropic salts which can inhibit many other processes (eg. PCR). Similar
reasoning for precaution in step 8.

8 Precaution: Flow-through must be discarded before this step or residual wash buffer will not
be completely removed.

9 -

10 Maximum elution efficiency achieved between pH 7.0 8.5, in low ionic strength buffer,
hence DI water can be used to achieve these conditions.

Restriction Digestion (Practical 1b)

Digesting a particular DNA molecule with a specific restriction enzyme results in generation of several
cleaved DNA fragments that may be fractionalized and subsequently studied or analysed.
[Refer to appendix for materials and procedure]

Step Explanation

1 The 10x RE Buffer contains a pH buffer, Mg (a needed cofactor) and salts, which are added
to provide an optimal environment (NaCl and/or MgCl2 concentration, pH) for the restriction
enzymes, Hind III and Bam HI to cleave at their specific restriction sites.
Plasmid DNA (pUCAg) is cleaved at the site of respective RE recognition sites present
within the molecule to isolate GFP gene from the plasmid DNA.
Restriction Enzymes are delicate and need to be treated carefully. Because enzymes are
proteins and proteins denature as the temperature is increased, the entire practical is
carried out on ice to slow down enzymes that can break it apart.

2 The tubes are pulse-spinned to collect the reaction mixture at the bottom of the tubes to
increase possibility of GFP gene insertion into pUCAg through complementary base pairing
at the restriction site.
 3
3 -

4 The tubes are then incubated at 37C as that is the optimal temperature at which the
restriction enzymes function to efficient restriction digestion.

5 -

Polymerase Chain Reaction, PCR (Practical 1c)

This process amplifies the gene of interest in order to prepare large amount of any specific DNA
segments within hours by allowing duplication of a specific piece of DNA repeatedly until several
millions of replica copies are obtained. This is especially useful for DNA manipulation where a large
amount of high quality DNA is needed.
[Refer to appendix for materials and procedure]

Step Explanation
1. The 10x PCR Buffer is added to provide a suitable environment for reagents (like Taq
Polymerase) to react with one another. MgCl2is added to function as a divalent cation which
is required by the Taq Polymerase to work. Primers 1 and 2 are needed in the anneal step
to attach to the 5 and 3 end of the single stranded DNA strands to allow extension of the
complementary DNA strand through the use of dNTPs, which are the ATCG nucleotides.
Taq Polymerase is a form of DNA Polymerase that catalyses the complementary base
pairing of the DNA strand and the dNTPs.
2. -
3. This step is performed to ensure that all the reagents are concentrated at the bottom of the
tube to increase chances of reagents reacting with one another to produce more PCR-
amplified products.
4. 94C (Melt): Heat breaks hydrogen bonds between DNA strands, hence denaturing double-
stranded DNA into two single strands. This allows a pair of oligonucleotide primers to attach
to the complementary sequences of the single-stranded template DNA in front of gene that
is to be replicated.
51C (Anneal): Allow primers to anneal to the template DNA.
72C (Extend): Taq Polymerase attach to 3 end of the primers annealed to the template
DNA. Optimum temperature at which Taq Polymerase is able to best catalyse the extension
of complementary DNA strand in 5 to 3 direction by addition of dNTPs.
Repeat: Once cycle is complete and new double-stranded DNA is formed, twice the original
amount of DNA can now be replicated by repeating above process. The cycle is repeated
25 times to produce 225 copies of the target DNA. After that, temperature is kept at 72C to
ensure that all single-stranded DNA become double stranded.
16C: Prevent any further reaction
5. -

Gel Electrophoresis (Practical 2)

Gel Electrophoresis allows identification and visualization of the DNA restriction fragments that
resulted after enzymatic digestion as well as the amplified GFP gene fragments
[Refer to appendix for materials and procedure]

Step Explanation
1. -
 4
2. Gel loading dye: Comprises of water, sucrose and a dye (Bromophenol Blue). The dye adds
colour to the sample for visual tracking of DNA migration during electrophoresis. The
presence of glycerol ensures that the DNA in the ladder and sample forms a layer at the
bottom of the well. It also increases density of the sample, ensuring that DNA drops evenly
into the well. EDTA is also included to chelate magnesium in enzymatic reactions, thereby
stopping the reaction.

3. Agarose Gel: Has large pore size and large range of separation, which makes it suitable for
electrophoresis of DNA fragments of usually 50-20,000 bp in size and large protein
molecules
TBE Buffer: TBE or Tris/Borate/EDTA, is a buffer solution containing a mixture of Tris base,
boric acid and EDTA. Tris-acid solutions are effective buffers for slightly basic conditions,
which cause the double-stranded DNA to denature and keep it in single-strand form which is
soluble in water and also confer negative charge that allows it to migrate to the positive
electrode in an applied electric field. EDTA protects the nucleic acids against enzymatic
degradation.

4. Since sugar-phosphate backbone of DNA confers a negative charge to the molecule, it will
move from towards the cathode. Shorter DNA fragments will move more quickly than longer
fragments because of the molecular sieving effects of agarose gel that exerts an opposite
force on migration of DNA fragment.

5. A nucleic acid stain is used to allow visualization of the DNA fragment upon excitation with
UV light before the restriction profile is captured or the presence of PCR is verified by a gel
documentation system.
Note: The gel apparatus should be connected to an electrical power supply with an appropriate
voltage to the gel. For mini gels, typical gradients used are between 15 volts/cm. Higher voltages
and shorter runs will decrease the resolution of the gel and may also cause overheating that may melt
the agarose.

Results
Well 1: 1k bp DNA ladder
Well 14: 100bp DNA ladder
Well 6-9: Results of HeeJun-YuHwa Pair
Well 7: Plasmid (7l)
Well 8: BamH I (12l)
Well 9: Hind III (12l)
Well 10: PCR (12l)
Well 10-13: Results of ShuHui-SzeMin Pair
Well 11: PCR (12l)
Well 12: Hind III (12l)
Well 13: BamH I (12l)
Well 14: Plasmid (7l)
Fig.1) Photograph of agarose gel by using gel documentation system 5

Distance migrated by bromophenol blue dye: 77mm

Table.1) Table of Relative mobility of DNA fragments of DNA ladder in well 1 and 14
DNA fragment length / bp Distance migrated by DNA Relative mobility (3 d.p.)
fragment / mm (1 d.p.)

1000 19.0 0.247

900 20.0 0.260

800 21.0 0.273

700 23.0 0.299

600 25.0 0.325

500 28.0 0.364

400 30.0 0.390

300 33.0 0.429

200 36.0 0.468

100 42.0 0.545

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90 45.0 0.584

80 47.0 0.610

70 49.0 0.636

60 51.0 0.662

50 53.0 0.688

40 56.0 0.727

30 59.0 0.766

20 62.0 0.805
10 66.0 0.857

Relative mobility of the GFP gene fragment from the Hind III digested sample of pUCAg
(HeeJun-YuHwa Pair): 19.5/77 = 0.253
(ShuHui-SzeMin Pair): 21.0/77 = 0.273
Referring to the graph below, the molecular size of the GFP gene fragment from the Hind III digested
sample of pUCAg can be estimated to be 1016 base pairs for HeeJun-YuHwa Pair and 880 base
pairs for ShuHui-SzeMin Pair. Since the value obtained by ShuHui-SzeMin Pair is closer to the
expected GFP gene size of 709 base pairs (including the PCR primers), it is more likely for this GFP
gene to be expressed upon completion of Practical 3.
Fig.2) Extrapolated graph of relative mobility of DNA fragments of DNA ladder against the molecular
size in base pairs (bp)

Extrapolated Graph of Relative mobility of DNA

fragments of the DNA ladder against the molecular
size, in base pairs (bp)
1
Relative mobility of DNA fragments of the DNA ladder

0.9
0.8
0.7
0.6
0.5
0.4
0.3 y = -0.14ln(x) + 1.2223
0.273
0.253
0.2
0.1
0
1016
0 200 400 600 800 880 1000 1200
molecular size / bp
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Fig.3) Semi-log Graph of Relative mobility of DNA fragments of the DNA ladder against the molecular
size, in base pairs (bp)

Semi-log Graph of Relative mobility of DNA fragments

of the DNA ladder against the molecular size, in base
pairs (bp)
1
Relative mobility of DNA fragments of the DNA ladder

0.9
0.8
0.7
0.6
0.5
0.4
0.3 y = -0.14ln(x) + 1.2223
0.273
0.253
0.2
0.1
0 880 1016
1 10 100 1000 10000
molecular size / bp

Heat Shock Transformation (Practical 3)

After the ligation to join the restriction fragments obtained through restriction endonuclease mediated
digestion of both vector and gene fragment of interest, forming recombinant plasmid carrying the
gene of interest. With the completed recombination, transformation allows the introduction of
recombinant plasmid DNAs into bacterial cells such as E.coli. The heat-shock used in this practical is
to create a thermal imbalance on either side of the cell membrane, which forces the DNA to enter the
cells through either cell pores or the damaged cell wall.
[Refer to appendix for materials and procedure]
Step Explanation
1. Competent cells: Cells that can bind and ingest exogenous high molecular weight DNA.
2.
3. Makes plasmid adhere to cell wall.
4. Reduces fluidity of cell membrane by releasing lipids and proteins.
Formation of pores on cell surface.
Depolarizes negatively-charged inner membrane, making it electrically neutral.
These facilitates the crossing of negatively-charged DNA through the cell membrane.
Also creates a thermal imbalance between internal and external environment of cell, helping
to pump DNA into the cell.
5. Closes pores, preventing plasmid from escaping.
Reduces thermal motion of DNA, promoting uptake of exogenous DNA.
6. Nutrients to encourage cellular growth.
7. Recover from treatment; grow, make DNA, RNA and proteins.
8. Separate bacterial cells and surrounding nutrient-depleted solution.
9. Remove nutrient-depleted solution.
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10. Screening. Since pUCAg contains ampicillin resistance gene while E. coli genome does not,
only successfully transformed E. coli cells that have taken up pUCAg will survive in the
ampicillin agar.
11. -

Results

Fig.4) Photo of successfully transformed bacterial Fig.5) Photo of successfully transformed bacterial
colonies (ShuHui-SzeMin Pair) colonies (ShuHui-SzeMin Pair) and unsuccessfully
transformed bacterial colonies (HeeJun-YuHwa Pair)
As predicted above, Sze Min-Shu Hui pair successfully carried out the heat shock transformation,
with bacterial colonies which glow with a greenish hue when exited with blue or UV light found on the
growth medium, while Hee Jun-Yu Hwa pairs plate did not show any sign of such colonies.

Discussion

Error Analysis
In general, many precautions were taken to ensure that we produced accurate results. For example,
use of gloves was emphasized for every experiment and pipette tips were discarded after each use.
For the gel electrophoresis, stabilisation of hands before loading our samples into the wells was
ensured so as to ensure that our setup would not be contaminated. However, due to the high difficulty
of some steps, the results produced may not have been accurate. This is especially the case for
Practical 1C (PCR) where 1l of reagents were required. Much difficulty was faced in pipetting such a
minute amount due to our lack of practice in pipetting. Unskilled pipetting might have led to the
withdrawal of incorrect reagent volume, affecting the PCR product. This could have caused the
inaccurate gel electrophoresis results, explaining the discrepancy of the approximated GFP gene
fragment which should have been 709 bp instead. Furthermore, the unsuccessful heat shock
transformation could be due to improper handling, where the bacteria was exposed to long periods of
room temperature between the heat shock and icing steps, reducing effects of extreme temperatures.
The process could have been further improved by being careful with the samples to ensure that there
is minimal contamination with foreign particles (ie. saliva when talking to each other). Being more
diligent and focused during experiment would further help in reducing possible human errors. These
experiments could be improved to yield more accurate results by doing repeats or replicates several
times.
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Subcloning GFP gene from pUCAg into expression vector
Preparation of pTRY and GFP gene from pUCAG*
*Assumption:
For this fabricated expression vector, pTRY, we are assuming that the chosen parent and destination
vector multiple cloning regions contain common restriction sites(two beside GFP gene in pUCAg and
one downstream a strong promoter in pTRY) that require common buffer, and neither of these
restriction sites occur within the insert.
With that assumption, we can use same two enzymes to digest the parent and destination vectors
with a common buffer**, followed by dephosphorylation of the destination vector in order to prevent
destination vector from self-ligating back to original plasmid(elaborated in the Appendix). The insert
and the dephosphorylated vector are then separated on an agarose gel and purified and ligated.
T4 DNA Ligase catalyzes the bond formation between the 5-PO4 of the insert and the 3-OH of the
vector. Dephosphorylating the vector prevents the second bond from forming in-vitro (indicated by the
OH). During transformation, these nicks are repaired in the bacteria.

Restriction Digestion of pUCAG and pTRY (Practical 1B)

Typically, transferring an insert from one vector to another needs digestion by two different REs (a
double digest). The reaction is simple if both restriction enzymes work well in same conditions. Simply
add 1l of the second restriction enzyme into the list of reagents (Appendix, Practical 1B) and adjust
the amount of water used. (It is important to note that restriction enzymes are commonly stabilized in
50% glycerol solution. Do not exceed 5% glycerol in final digest with the two enzymes. Glycerol
concentrations of more than 5% may lead to star activity.)
A type II restriction enzyme is used, which will cleave the double stranded DNA at its specfic
restriction sites, regions of palindromic nucleotide sequences, which, when cleaved, will form
overhanging sticky ends. This will allow precise excision of the GFP gene with no interference of the
gene.
Using the same restriction enzymes that have been used for restriction digestion of pUCAg, digest
the pTRY plasmid and according to the assumption made, after the digestion, there will be a single
cut in its multiple cloning region (MCR) beside a promoter. Similar to restriction digestion of pUCAg,
the restriction enzymes will cut the vector to form sticky ends and since it is cut by the same enzymes,
those sticky ends formed will be complementary to that of the sticky ends of GFP gene.

Dephosphorylating Vectors to Limit Self-Ligation

Preventing vector self-ligation is critical for reducing subcloning background. The efficiency of ligating
the plasmid to itself is far better than ligating a separate piece of DNA into the vector and is the
favoured reaction. Removing the 5 phosphates of the linearized vector will prevent T4 DNA Ligase
from recircularizing the vector. After dephosphorylation, the enzyme must be removed either by direct
purification or gel electrophoresis and gel isolation with DNA purification as if not, the GFP gene itself
will not be able to bind to the restriction site.

Gel Isolation (Practical 2)

Purification of the insert and destination vector is required to improve the efficiency of subcloning by
segregating the wanted reactants from the unwanted reactants. This reduces background by
eliminating uncut vector from transformation. Therefore, once all the vectors and insert is cut, gel
electrophoresis like the one described in Practical 2 is carried out, separating DNA fragments of
different base pair sizes into different bands. Identify and cut the band of interest for each cut pTRY
vector and insert by comparing the expected and actual results with DNA ladder. It is possible to
isolate the band containing the necessary DNA fragments and extract it from the agarose gel using a
razor blade, making it ready for the next step of sub-cloning, ligation.
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Polymerase Chain Reaction (Practical 1c)
PCR amplifies the gene fragments of GFP gene and pTRY, prepared in previous steps, exponentially
to obtain sufficient quantity for subsequent cloning expleriments. This involves rapid cycling of
temperature in a PCR machine that achieves 94C for melts, denaturation of double-stranded
template into a single strand, 50-55C for annealing of oligonucleotide primers to the single strand,
and 72-74C for addition of complementary nucleotide bases by Taq polymerase.

Ligation of Vector and Insert

DNA ligase catalyzes phosphodiester bond formation between adjacent nucleotides by hydrolyzing
ATP. Ligating amplified pTRY plasmid and GFP gene in vitro should be carried out under stable
conditions of heat, acidity and glycerol concentrations. Complementary sticky ends on pTRY and
GFP insert will complementary base pair, making it easy for DNA ligase to form the covalent bond.
Adding an excess of GFP gene ensures most recombinant pTRY contain the GFP gene, increasing
the success rate.

Heat Shock Transformation (Practical 3)

After ligation, the recombinant plasmid DNAs are ready for introduction into the E.coli host, as most
species of bacteria are able to take up DNA molecules from the medium in which they grow. Under
laboratory conditions, to better facilitate this uptake of DNA, bacterial cells are grown to mid-long
phase before harvesting and treating them with solution containing a divalent cation to render them
competent to foreign DNA. The recombinant plasmid DNA and bacteria are then mixed together in
the same solution and heat-shock transformation steps described above is then applied. This is
followed by a recovery step which involves the culture of the treated bacterial cells in LB liquid broth.
These treatments are necessarily harsh and most bacterial cells do not survive but they renders
those surviving cell wall permeable and it becomes capable of picking up foreign DNA.

Screening for Recombinants

After the transformation, to select for the transformed cells of interest, the transformed bacterial cells
are plated on a cell culture containing antibiotics or other selective agents according to components
of pTRY. Those cells that did not take up recombinant plasmids that contain necessary agents that
allows it to survive in extreme conditions die upon exposure to the agents added on the cell culture.
By viewing the E.coli cell colonies under UV light, it is possible to determine which colonies have
taken in the recombinant pTRY containing GFP gene. These colonies would have expressed the
gene, producing green fluorescence protein which result in the colonies showing bright green
fluorescence under UV light. These colonies are successful subclones of GFP from pUCAg into pTRY
and can be used for further study.

Conclusion
In conclusion, in this practical, we were able to identify and visualize the GFP gene through isolation
and restriction digestion of pUCAg, followed by Polymerase Chain Reaction and gel electrophoresis
for further analysis of the DNA fragments. This ultimately showed that the approximate size of the
GFP gene according to the gel electrophoresis result is about 880 base pairs, despite the disparity
between expected value of 709 bp and actual result which might have been caused by human errors
in Practical 1 and 2 steps. And lastly, after practical 3, we successfully identified actual bacterial
colonies with recombinant plasmid that allows expression of GFP gene through viewing the growth
plate under blue light. In this report, we have also evaluated the purposes of each step and reagent in
the practical procedures and discussed the possible reasons that could have led to our inaccurate
data. Method of sub-cloning of GFP gene from pUCAg into an expression vector, pTRY, was also
explained in detailed steps, in the discussion corner to show an application of the procedures
described in practical 1, 2 and 3. All in all, as DNA sequencing methods is essential in studying
molecular genetics, it is beneficial that we are able to try our hands on them.
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Appendix
Isolation of Plasmid DNA (Practical 1a)
Materials
Buffer P1 (50mM Tris-HCl pH 8.0, 10mM EDTA, 100g/ml RNaseA)
Buffer P2 (200mM NaOH, 1% SDS)
Buffer N3 (Guanidine hydrochloride, potassium acetate, pH 4.8)
Buffer PB (Guanidine hydrochloride, 30% isopropanol)
Buffer PE (10mM Tris-Cl, pH 7.5, 80% ethanol)
Sterile distilled water
Tabletop centrifuge machine
Sterile 1.5ml microcentrifuge tubes

Procedure
1. Resuspend 5 ml of pelleted bacterial cells in 250 l Buffer P1 in a microcentrifuge tube. Note:
No cell clumps should be visible after resuspension of the pellet.
2. Add 250 l Buffer P2 and mix thoroughly by inverting the tube 4-6 times, or until solution
becomes viscous and slightly clear. Precaution: Do not vortex; do not allow lysis reaction to
proceed for more than 5 min.
3. Add 350 l Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times. The
solution should become cloudy.
4. Centrifuge for ten min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge. A compact
white pellet will form.
5. Apply the supernatants from step 4 to the QIAprep spin column by decanting or pipetting.
Centrifuge for 30s. Discard the flow-through.
6. Wash QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 10s. Discard the
flow-through.
7. Wash QIAprep spin column by adding 0.75 Buffer PE and centrifuging for 10s. Discard the
flow-through.
8. Centrifuge for an additional 1 min to remove residual wash buffer.
9. Place QIAprep column in clean 1.5 ml microcentrifuge tube.
10. To elute DNA, add 50 l dH2O to the center of each QIAprep spin column, let stand for 1 min
and centrifuge for 1 min.

Restriction Digestion (Practical 1b)

Materials
pUCAg
Sterile 1.5ml microcentrifuge tubes
10x RE Buffers
Restriction Enzymes - Hind III and BamH I
Tabletop Centrifuge Machine
37C Water Bath

Procedure
1. Label, and set up on ice, microcentrifuge tubes as described below. Add the reagents in the
order given, using the white pipette and white tips.
Reagents Reaction 1 (Hind III) Reaction 2 (BamH I)

10X RE BUffer 1l 1l
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Plasmid DNA 8.5l 8.5l
(pUCAg)

Restriction Enzyme 0.5l 0.5l

Total Volume 10l 10l

2. Cap the tubes and tap them a few times to mix the reagents together.
3. Pulse-spins the tubes to collect the reaction mixture to the bottom of the tubes.
4. Incubate the tubes at 37C for 60 minutes in a water bath.
5. Store the tubes at -20C.

Polymerase Chain Reaction (Practical 1c)

Materials
10x PCR Buffer
Tabletop centrifuge machine
dNTPs mix (containing dATP, dTTP, dGTP, dCTP)
Thin Walled PCR Tube
Primers (Pgfp1 and Pgfp2)
Sterile 1.5ml microcentrifuge tube
25 mM MgCl2
Sterile distilled water
DNA template (Isolated plasmid, pUCAg)
Taq Polymerase
PCR Machine

Procedure
1. Prepare the following reaction mixture in a thin walled PCR tube. Reagents must be added in
the following order:
ddH2O 37l
10x PCR Buffer 5l
25mM MgCl2 3l
Primer 1 (Pgfp1) 1l
Primer 2 (Pgfp2) 1l
DNA (pUCAg) 1l
dNTPS 1l
Taq Polymerase (1U) 1l
TOTAL 50l

2. Mix the various components well, by gentle vortexing or pipetting the mixture up and down a
few times.
3. Pulse-spin PCR tube to collect content to the bottom of the tube.
4. Place PCR tube into PCR machine pre-set with the following cycling parameters.
a. 2 min 94C
b. 30 sec 94C
c. 30 sec 51C
d. 1 min 72C
e. 10 min 72C
f. 15 min 16C
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Repeat steps b, c and d for 25 cycles.

Gel Electrophoresis (Practical 2)

Materials
BamHI and HindIII digested UCAg
Tabletop centrifuge machine
6x gel loading dye
TBE Buffer
Agarose Gel
PCR product

Procedure
1. Thaw the DNA samples from Practical 2. This includes the BamHI and HindIII digested
plasmid samples as well as the amplified GFP gene samples
2. Add appropriate volume of gel loading dye into each sample and tap each tube to mix
thoroughly. Pulse-spins the tubes to collect the samples at the bottom of the tubes.
3. Load the content of each sample tube into a sample well in the prepared agarose gel (placed
in the electrophoresis apparatus and immersed in TBE buffer) carefully without spilling over
into the neighbouring wells
4. Replace the cover of the electrophoresis apparatus and then electrophorese at about 100 volts
for 20 to 30 minutes or until the bromophenol blue dye (dark blue colour) has migrated about
two-third the length of the agarose gel
5. Take a photograph of the gel by using the gel documentation system and use the following
DNA ladders to help check the sizes of the DNA fragments of your samples

Heat Shock Transformation (Practical 3)

Materials
Competent DH5 cells
pUCAg
Sterile 1.5-ml microcentrifuge tubes
Bacterial culture tubes
250-ml beaker half filled with ice
42C water bath
Tabletop centrifuge machine
LB Broth
LB agar plate supplemented with ampicillin
Glass spreader

Procedure
1. Thaw a tube of competent cells on ice.
2. Swirl or tap the tube to gently mix the cells after thawing.
3. Add 1 l of pUCAg to the tube of competent cells and incubate on ice for 30 min.
4. Heat shock the cells by immersing the tube in a 42C water bath for 90 sec.
5. Incubate the tube on ice for 2 min.
6. Add 1 ml of LB medium without any antibiotics into the tube.
7. Incubate the culture tube in a 37C incubator with shaking for 45 min.
8. Centrifuge the culture tube at 10,000 rpm for 1 min.
9. Remove most of the supernatant until approximately 50 to 100 l of the culture medium is left,
before re-suspending the bacterial cell pellet.
10. Transfer the bacterial suspension onto an LB plate supplemented with ampicillin and spread
the suspension evenly across the plate with a sterile glass spreader
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11. Incubate the plate overnight in a 37C incubator and bacterial cells transformed with the
plasmid DNA will form single colonies.

Discussion
Choosing the right Restriction Endonucleases
Restriction enzymes or restriction endonucleases (RE) identify short, frequently palindromic DNA
sequences (called restriction sites) that are specific to the RE. Type II REs cleave double-stranded
DNA (dsDNA). Hence, they are selected to cleave the plasmids, which are also double-stranded, in or
beside their restriction sites.
Depending on the restriction enzyme sites available on the plasmid, around and inside gene of
interest, and varied optimal condition for each of the restriction enzymes, the reaction and protocol for
restriction digestion step varies.
Therefore, first step of choosing restriction enzyme is to determine restriction sites of the source and
destination plasmid in order to find an appropriate enzyme that has restriction sites on both plasmids:
there must be at least 2 sites that fall near the gene of interest but not in the gene itself for source
plasmid, while for the destination plasmid, there must be only one restriction site that is compatible
with the restriction enzyme used to cut the gene of interest.

Even after finding common restriction enzymes that meets the criteria above, each RE's specific
requirements for optimal activity has to be taken into consideration.
Each restriction enzyme has its own combination of optimal conditions which result in the most
activity and accuracy for its function. These include, but are not exhaustive of temperature, pH,
enzyme cofactors, salt composition and ionic strength.
The optimal temperature for most REs is 37C, but depending on the REs origin, they may have
higher or lower optimal temperatures. For instance, Bst XI and Bst Z, isolated from thermophilic
bacterial, work best at 50C while Sma I works best at 25C.

If REs with two different working temperatures are to be used, the digestion requiring the lower
temperature should be performed first, followed by that for the higher temperature (sequential digest
method). For enzymes that require temperatures above 37C, star activity may occur due to
evaporation, which leads to higher glycerol concentration.

Another situation is when the enzymes require different reaction buffers. A buffer in which both
enzymes has at least 75% activity is ideal since both enzymes have acceptable activity levels in this
buffer.

Star Activity
Restriction enzymes, under nonstandard conditions, can demonstrate the ability to cleave DNA at
sequences different from their defined recognition sites. The term star activity has been given to this
nonsequence-specific cleavage of DNA under non-optimal reaction conditions. The most common
types of altered activity are single-base substitutions, truncation of the outer bases in the recognition
sequence and single-strand nicking. In general, star activity is not a concern if restriction
endonucleases are used in the recommended buffers at the appropriate temperatures. Star activity is
evident with a number of restriction enzymes when the following parameters are altered in the
reaction environment:
High enzyme concentration (generally >100 units/g).
High glycerol content (>5% v/v).
Substitution of Mn2+ for Mg2+ (or substitution of other divalent cations).
Low salt concentration (generally <25mM).
Extremes of pH, especially pH>8.0.
Presence of DMSO, ethanol or other organic solvents.
 15

Complete sequence of shuttle vector pUCAg.Gfp:

ORIGIN
1 caggaaacag ctatgaccat gattacgcca agcttgttta aacatttaaa tctcgagcca
61 tggattcgac attgattatt gactagttat taatagtaat caattacggg gtcattagtt
121 catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga
181 ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca
241 atagggactt tccattgacg tcaatgggtg gactatttac ggtaaactgc ccacttggca
301 gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg
361 cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc
421 tacgtattag tcatcgctat taccatgggt cgaggtgagc cccacgttct gcttcactct
481 ccccatctcc cccccctccc cacccccaat tttgtattta tttatttttt aattattttg
541 tgcagcgatg ggggcggggg gggggggggc gcgcgccagg cggggcgggg cggggcgagg
601 ggcggggcgg ggcgaggcgg agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa
661 agtttccttt tatggcgagg cggcggcggc ggcggcccta taaaaagcga agcgcgcggc
721 gggcgggagt cgctgcgttg ccttcgcccc gtgccccgct ccgcgccgcc tcgcgccgcc
781 cgccccggct ctgactgacc gcgttactcc cacaggtgag cgggcgggac ggcccttctc
841 ctccgggctg taattagcgc ttggtttaat gacggctcgt ttcttttctg tggctgcgtg
901 aaagccttaa agggctccgg gagggccctt tgtgcggggg ggagcggctc ggggggtgcg
961 tgcgtgtgtg tgtgcgtggg gagcgccgcg tgcggcccgc gctgcccggc ggctgtgagc
1021 gctgcgggcg cggcgcgggg ctttgtgcgc tccgcgtgtg cgcgagggga gcgcggccgg
1081 gggcggtgcc ccgcggtgcg ggggggctgc gaggggaaca aaggctgcgt gcggggtgtg
1141 tgcgtggggg ggtgagcagg gggtgtgggc gcggcggtcg ggctgtaacc cccccctgca
1201 cccccctccc cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtgcggggcg
1261 tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc cgggcggggc
1321 ggggccgcct cgggccgggg agggctcggg ggaggggcgc ggcggccccg gagcgccggc
1381 ggctgtcgag gcgcggcgag ccgcagccat tgccttttat ggtaatcgtg cgagagggcg
1441 cagggacttc ctttgtccca aatctggcgg agccgaaatc tgggaggcgc cgccgcaccc
1501 cctctagcgg gcgcgggcga agcggtgcgg cgccggcagg aaggaaatgg gcggggaggg
1561 ccttcgtgcg tcgccgcgcc gccgtcccct tctccatctc cagcctcggg gctgccgcag
1621 ggggacggct gccttcgggg gggacggggc agggcggggt tcggcttctg gcgtgtgacc
1681 ggcggctcta gagcctctgc taaccatgtt catgccttct tctttttcct acagctcctg
1741 ggcaacgtgc tggttgttgt gctgtctcat cattttggca aagaattatc gcatgcctgc
1801 agagctctag agtcgacgaa ttcaccatgg tgagcaagca gatcctgaag aacaccggcc
1861 tgcaggagat catgagcttc aaggtgaacc tggagggcgt ggtgaacaac cacgtgttca
1921 ccatggaggg ctgcggcaag ggcaacatcc tgttcggcaa ccagctggtg cagatccgcg
1981 tgaccaaggg cgcccccctg cccttcgcct tcgacatcct gagccccgcc ttccagtacg
2041 gcaaccgcac cttcaccaag taccccgagg acatcagcga cttcttcatc cagagcttcc
2101 ccgccggctt cgtgtacgag cgcaccctgc gctacgagga cggcggcctg gtggagatcc
2161 gcagcgacat caacctgatc gaggagatgt tcgtgtaccg cgtggagtac aagggccgca
2221 acttccccaa cgacggcccc gtgatgaaga agaccatcac cggcctgcag cccagcttcg
 16
2281 aggtggtgta catgaacgac ggcgtgctgg tgggccaggt gatcctggtg taccgcctga
2341 acagcggcaa gttctacagc tgccacatgc gcaccctgat gaagagcaag ggcgtggtga
2401 aggacttccc cgagtaccac ttcatccagc accgcctgga gaagacctac gtggaggacg
2461 gcggcttcgt ggagcagcac gagaccgcca tcgcccagct gaccagcctg ggcaagcccc
2521 tgggcagcct gcacgagtgg gtgtaatagc tcgagcggcc gcacttaagt tacgcgtgga
2581 tcaattcact cctcaggtgc aggctgccta tcagaaggtg gtggctggtg tggccaatgc
2641 cctggctcac aaataccact gagatctttt tccctctgcc aaaaattatg gggacatcat
2701 gaagcccctt gagcatctga cttctggcta ataaaggaaa tttattttca ttgcaatagt
2761 gtgttggaat tttttgtgtc tctcactcgg aaggacatat gggagggcaa atcatttaaa
2821 acatcagaat gagtatttgg tttagagttt ggcaacatat gccatatgct ggctgccatg
2881 aacaaaggtg gctataaaga ggtcatcagt atatgaaaca gccccctgct gtccattcct
2941 tattccatag aaaagccttg acttgaggtt agattttttt tatattttgt tttgtgttat
3001 ttttttcttt aacatcccta aaattttcct tacatgtttt actagccaga tttttcctcc
3061 tctcctgact actcccagtc atagctgtcc ctcttctctt atgaagatcc ctcgacctgc
3121 agcccaagct gatcccggga tttaaatgtt taaacgaatt cactggccgt cgttttacaa
3181 cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct
3241 ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc
3301 agcctgaatg gcgaatggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
3361 tcacaccgca tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
3421 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
3481 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
3541 tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc
3601 atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc
3661 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
3721 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
3781 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
3841 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
3901 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
3961 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
4021 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
4081 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
4141 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
4201 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
4261 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
4321 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
4381 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
4441 attgctgata aatctggagc ccgtgagcgt gggtctcgcg gtatcattgc agcactgggg
4501 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
4561 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
4621 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
4681 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
4741 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
 17
4801 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
4861 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
4921 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta
4981 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
5041 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
5101 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
5161 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
5221 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
5281 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
5341 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
5401 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
5461 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
5521 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct
5581 ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa
5641 gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct
5701 ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac
5761 a

CCATGG BamHI restriction site

AAGCTT HindIII restriction site

References
http://abe.leeward.hawaii.edu/Protocols/QiagenSpinprepProtocol.htm
http://www.ncbi.nlm.nih.gov/nuccore/122893037
http://www.chem.uky.edu/courses/che554/Review_MolBio_30Apr2012.pdf
http://www.qiagen.com/products/catalog/sample-technologies/dna-sample-technologies/plasmid-
dna/qiaprep-spin-miniprep-columns
http://sbio.uct.ac.za/wp-content/uploads/2013/02/cloning_promega.pdf
http://amrita.vlab.co.in/?sub=3&brch=77&sim=314&cnt=1
http://www.symposcium.com/2012/07/why-heat-shock-incubate-on-ice-transformation/
http://www.madsci.org/posts/archives/2008-09/1220301763.Cb.r.html
https://www.umassmed.edu/uploadedFiles/trans%20bact%20cells(1).pdf
http://www.protocol-online.org/biology-forums/posts/38590.html
http://www.protocol-online.org/biology-forums/posts/8364.html
http://bio.classes.ucsc.edu/bio105l/EXERCISES/PCR%20cleanup.pdf
http://www.picse.net/CD2011/dna/mechanismOfPCR.html
http://gmo-crl.jrc.ec.europa.eu/capacitybuilding/manuals/Manual%20EN/Session05.pdf
http://wiki.answers.com/Q/Why_does_DNA_separate_in_agarose_gel_in_electrophoresis?#slide=15