OBJECTIVES 1. To amplify DNA using PCR 2.

To prepare agarose gel and analyze PCR product

DISCUSSION In this experiment, we had conducted PCR amplification of gene from plasmid DNA, also analysing PCR products by using agarose gel electrophoresis. The results were shown in figure 1. In PCR amplification of gene of interest, Taq polymerase was used. This enzyme has low fidelity, no 3-5 exonuclease activity (no proofreading activity) which can cause mismatches. 5u/l Taq polymerase was used as prevention for any inhibitors present in the reaction mixture, for example, the template DNA used was not highly purified. Besides, this amount of Taq polymerase was helpful in obtaining a better yield of amplification products. The total number of PCR cycles done was 35 times, it means that the initial quantity of template DNA was high. The number of PCR cycles depends on the amount of template DNA in the reaction mixture and the expected yield of the PCR product. In analysing PCR products by using agarose gel electrophoresis, 1% gel was used because it will shows good resolution of DNA fragments. The plasmid DNA fragments were separated based on their molecular weight, the lower molecular weight fragments will migrate faster that the higher molecular weight. Besides that, DNA fragments were separated based on charge, negatively charged DNA pulled toward the positive field of the gel. Agarose gel provides matrix with pores to allow DNA to travel through. The smaller fragments move faster, larger fragments will move slower. During the experiment, lane 9 was loaded with master mix without the DNA template, and this was called a negative control to observe if there was any primer dimer formed. If there was primer dimer present, bands or smearing of moderate to high intensity at position 40-50 base pairs can be observed. Through the results obtained, there was no primer dimer formed which means that the product was pure. Based on the result obtained, only single thick band was observed with molecular weight of 500bp. It shows that the overall PCR process was a success, with high DNA concentration, no contamination and without the presence of primer dimer.
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CONCLUSION In this experiment we had conducted PCR amplification of gene from plasmid DNA. The product of PCR had been analyze from the result obtain through agarose gel electrophoresis. From the result, it was concluded that no primer dimer was obtain and the band was in high concentration with no contamination. REFERENCES 1. http://2012.igem.org/Team:Goettingen/Project/Methods 2. http://projects.nfstc.org/workshops/resources/articles/A%20Systematic%20Analysis% 20of%20PCR%20Contamination.pdf 3. http://paws.wcu.edu/michaelis/Lab%20PCR%20and%20gel%20electrophoresis.pdf