PCR_amplification

PCR_amplification - PCR Amplification To amplify a particular sequence from some DNA source you must start with a template appropriate 5 and 3

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PCR Amplification February 21, 2009 To amplify a particular sequence from some DNA source, you must start with a template, appropriate 5’ and 3’ primer oligonucleotides, dNTPS, and a source of thermostable DNA polymerase with its accompanying buffer (usually Taq pol). PCR cycles through the processes of denaturing the DNA, annealing the primers, and extending the primers (DNA synthesis) multiple times (usually 30X). This allows for an exponential amplification of the DNA sequence found between the primers. Variables that must be considered include -- 1. Source of template: Are you using genomic DNA, plasmid DNA, a PCR product itself? Genomic DNA has the most complexity (your gene of interest is a very small fraction of the total DNA in the sample) and is therefore the most difficult to amplify. Don’t worry, this is only sometimes a problem. 2. Length of PCR target sequence: Short sequences (< 1KB) are usually simple to amplify and shouldn’t need more than 1 minute extension times. The longer it gets, the more
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This note was uploaded on 11/25/2009 for the course BI 379 taught by Professor Kavaler during the Spring '09 term at Colby.

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PCR_amplification - PCR Amplification To amplify a particular sequence from some DNA source you must start with a template appropriate 5 and 3

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