Polymerase Chain Reaction

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Titles: Amplification of lacZ gene by the Polymerase Chain Reaction Aim: To amplify the number of copies of lacZ gene fragment via Polymerase chain reaction from the plasmid pRY121 DNA. Abstract: The lacZ gene was amplified from 1 ng of plasmid pRY121 DNA using synthetic oligonucleotide primers A and B and Taq polymerase in 30 PCR cycles of 96˚, 55˚, and 72˚C in a DNA thermocycler. Agarose gel electrophoresis of BamH-restricted, mini-preped plasmid pRY121 DNA confirmed that lacZ gene fragments were produced. The mass and kb size of the amplified DNA was estimated at 147 ng and 0.6 kb respectively from comparisons to a 1 kb DNA ladder. Procedure: See pages 59-61of Reference [1] for the methodology used. Amendments: See Table 1 for changes made to volumes of reagents and materials used in Exercise 7: Table1. Corrective volumetric Table for PCR reaction mixture ______________ * With the 2.75 µL of plasmid DNA used made from a 1:1000 dilution of the mini-prep DNA (exercise 4-5); see calculations The 2 controls, 1 positive and 1 negative for DNA, tests the reliability of the procedure performed in general and to detect for the presence of any exo-plasmid DNA contamination in the reaction mixture. Calculations: From Expt. 4-5 A (260 nm)A(280 nm) = 0.3630.175=2.07 For pure RNA: A (260 nm)A (280 nm)≡2.0 ∴ Pellet was almost completely RNA AsA (260 nm)A(280 nm)→1.8, A260 nm can be used to estimate the mass of DNA present Given A260 nm (50 ng µL-1) =1 ∴ m (DNA) = 50 ng X 0.363 = 18.15 ng Volume of solution analysed by U.V. spectrophotometry: 1.0 µL ∴ CDNA = 18.15 ng µL-1 Discussion: E.coli β-galactosidase, encoded for by the lacZ gene, consist of 1024 amino acids [3], therefore lacZ should a minimum codon sequence of about approximately 3.07 kb. BamH1 has one restriction recognition site on the plasmid pRY121 located at the 2780th

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