Molecular Biology 300817: PCR Lab Report, Site-directed mutagenesis

This PCR lab report details an experiment focused on introducing an R204Q point mutation into TP63 cDNA through PCR reactions. The experiment involved testing primer pairs at different annealing temperatures, ligating the purified PCR amplicon into a plasmid, and transforming the result into competent E. coli for cloning. Furthermore, it aimed to digest the purified plasmid using a restriction endonuclease enzyme. The report outlines the procedures for each module, including gel electrophoresis and dilution techniques, along with the results obtained. Despite some initial issues with the results in module 2, the experiment proceeded to successfully synthesize amplicons, determine optimal concentrations, and analyze the size of the amplicons and plasmids. The report concludes that, despite a gel breaking, the objectives of the experiment were successfully met, demonstrating the successful implementation of site-directed mutagenesis and cloning techniques.

PCR Lab Report
Name of Student
Name of Professor
Date

Aims
The main goal of the experiment was to introduce R204Q point mutation into TP63 cDNA
through both the forward and reverse PCR reactions. The two primer pairs were tested at three
different annealing temperature. The experiment also seeks to ligate purified PCR amplicon into
plasmid pCR2.1 and the result transferred into competent E coli bacteria plate for cloning. It also
aims to try the digestion of the purified plasmid by using restriction endonuclease enzyme.
Procedure
Procedure for module 2
50 mL of the 1x TBE stock was used to prepare 1.2% weight to run the PCR samples. The DNA
band on the agarose gel was stained using 5uL of nucleic acid. 5 uL of the hyper ladder was
loaded in the first lane. Prime pair 1 (TP63_556 forward and TP63_R204Q_reverse) was loaded
into lane 2, 3 and 4. The prime pair 2 (forward and TP63 and TP63_1284 reverse) was loaded
into lane 5, 6 and 7. cDNA was loaded into lane 8. Also, 2 uL of loading dye was added to the 10
uL DNA samples including the cDNA to track the DNA fragments on the gel. The agarose gel
ran for 35 minutes at 110V with the yellow band being tracked for halfway run.
Procedure for module 3
Each amplicon used in module 2 was combined to make the TP63 amplicon in full length and
containing the R204Q point mutation. The tubes with the highest number of amplicons and also
having the correct sizes were selected. 1:10 dilution was then prepared by removing 5 uL from
each of the selected tubes and replaced by 90 uL of sterile water. The two tubes were then
combined into a 1.5 tube. From the 1:10 dilution, 1:100 and 1:1000 were made and then tested
using annealing temperatures 64 and 60. The gel agarose was prepared in the same way as in
module 2. Lane 2 and 3 had PCR reaction with dilution factor 1:10. Lane 4 and 5 had a dilution
factor of 1:100 while 6 and 7 had a dilution factor f 1:1000.
Procedure for module 4
This involved performing the digestion of the purified plasmid by using restriction endonuclease
enzyme using 7.5 uL of the purified plasmid or purified TP63 amplicon. Four 1.5 mL tubes were
needed. In the first purified plasmid tube, water was added as a control. Bts-1 water mix was
added as a test in the second tube. The same pattern was followed for the third and fourth tubes.
2 uL of 10x restriction endonuclease buffer was added to all the four tubes. The tubes were then
taken for incubation at 55 degrees Celsius for thirty to forty minutes. During this time, 1.2 %
agarose gel was prepared and 50 mL of 1x TBE added. O.6g of agarose and nucleic acid were
also added for the DNA gel stain. 10 uL from the restriction digest tubes and 2 uL of DNA
loading dye were taken and mixed together to be loaded into gel walls. The same pattern was
followed for gel 1 with hyper ladder loaded in lane 1, lane 2 consisted of purified plasmid with
water, lane 3 with purified plasmid treated with restriction endonuclease Bts1. Lane 4 had
purified amplicon with only water and lane 5 had purified amplicon treated with the restriction
endonuclease. The cDNA sample was not run in this module.

Results

Lane 1 2 3 4 5 6 7 8
Figure 1: Gel results for module 2
The results found for module 2 were wrong so new samples were given and later used for the
experiment.

Ladder
Lane 1 2 3 4 5 6 7
Figure 2: Gel results for module 3

Lane 1 2 3 4 5
Figure 3: Gel results for module 4
Conclusion
For module 2, synthesis of two TP63 amplicons was done to introduce an R204Q point mutation
into a TP63 cDNA. The results in this module were wrong due to unclear reasons. As a result,
the example gel was used. The expected size of primer 1 was 267 and that for primer 2 was 467.

According to the hyper ladder for primer 1 it shows to be between 200 and 400 and primer 2
between 400 and 600.
Three different dilutions were tested in module 3. Two different temperatures were used to
determine the concentration with the highest number and correct size of the amplicons. This size
should be 729 bp according to computer class. The size of the amplicons was seen to be between
600 and 800 which is correct.
Theoretically, for module 4 and using computer class the size of the plasmid with insertion
should be 4698 bp and without insertion it should be 3929 bp. From the results, the size of the
purified plasmid was near the expected value. Even though the gel broke before samples could
be run, the objectives of the experiment were successfully met.