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PCR_amplification_2

PCR_amplification_2 - μ M 1.0 μ l of 3’ primer at 20 μ...

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PCR Amplification 2 February 26, 2009 For difficult PCRs, I now typically use the Fail Safe PCR kit from Epicentre (see this link ). This includes a set of 12 buffers at 2X concentration (Buffers A – L) that all contain the appropriate salt and buffer conditions as well as all the dNTPs. Each buffer varies in the amount of MgCl2 (increasing concentration from A to L) and the amount of the special PCR “enhancer” that Epicentre makes (also increases from A to L). The kit also contains a mixture of two thermostable enzymes that allow for higher fidelity PCR. The enzyme mix is about ten times as expensive as regular Taq polymerase so I usually just use regular Taq unless fidelity is essential. Set-up for "Typical" Failsafe PCR amplification: Thaw all reagents, mix well and place on ice. For a 50 µl reaction, place these reagents in a 200 μ l PCR tube: 21.8 μ l of H 2 O 25.0 μ l of 2X PCR buffer (A-L) 1.0 μ l of 5’ primer at 20
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Unformatted text preview: μ M. 1.0 μ l of 3’ primer at 20 μ M. 1.0 μ l of DNA template (usually 10-100 ng of total DNA) .2 ml of PCR enzyme (Taq polymerase) or about 1 unit 50.0 μ l total volume Cycle Program for "Typical" PCR amplification: Place these reagents in a 200 μ l PCR tube: 1. 2:00 minutes at 95ºC (to denature the original template) – this is a one-time step! 2. 0:20 seconds at 95ºC (to denature each successive amplification product) 3. 0:20 seconds at 55ºC (to allow the primers to anneal) The annealing temperature will vary depending upon your primer selection. 4. 2:00 minute at 72º (DNA synthesis) a good rule of thumb is to have 1 minute for every 1000 bases you need to synthesize 5. Cycle steps 2-4 for 30 times. 6. 10:00 minutes at 72ºC (allows the enzyme to complete any “unfinished” strands) 7. 10ºC “for ever” (store cold until ready for analysis)...
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