Polymerase Chain Reaction

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Lab 8: Polymerase Chain Reaction

Introduction: Polymerase Chain Reaction is based on the ability of DNA polymerase to symthesize new strand of DNA complementary to the offered template strand. It is a technique used to amplify a single or few copies of a piece of DNA across several orders of magnitude generating thousands to millions of copies of a particular DNA sequence. This lab will be used to determine how many copies and how far a particular piece of DNA migrates and generates.

Materials and Methods: Puc19 plasmid DNA will be our template and we will amplify the bla gene using Primers C and D. Also we will amplify a product of 1.3 kb using Primers A and B.

1. Label 2 PCR tubes “1” and “2”.

2. Add 18.5 ul of sterile water into each tube.

3. Add 0.5 ul of template pUC 19 to each tube.

4. Add 1.0 ul of Primers A and B to tube “1”.

5. Add 1.0 ul of primers C and D to tube “2”.

6. Add 20 ul of PCR mix to each tube, mix Gently. Place tube in the PCR machine and run.

Results: PCR is valuable because it allows the multiplication of unique regions of DNA so they can be detected in large genomes. The Taq polymerase begins adding nucleotides to the primer and eventually makes a complementary copy of the template. Each newly synthesized DNA piece can act as a new template, so after 30 cycles, millions of copies of a single piece of DNA can be produced.

Discussion: We have yet to view the DNA bands. But, as a result of PCR many copies are to be expected. If the strands are not sufficient perhaps our temperatures may have had a huge factor in the outcome.

References: C. I. Kado and S.-T Liu, 1981, Rapid Procedure for Detection and Isolation of Small and Large Plasmids, Journal of Bacteriology, Vol. 145, No. 3, p 1365-1373.

Paul Rabinow, 1996, Makin PCR A Story of Biotechnology, The University of Chicago Press,…...

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