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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.
Step Explanation
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
2
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.
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 -
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. -
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
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.)
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)
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
7
Fig.3) Semi-log Graph of Relative mobility of DNA fragments of the DNA ladder against the molecular
size, in base pairs (bp)
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
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.
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.
11
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.
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)
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.
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
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
14
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
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