Technique / Molecular Biology / PCR / Overlapping PCR

Overlapping PCR procedure

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	By Elsie on 29-Feb-2008 I'm gong to do the overlap pcr and I have designed two completely complementary primers but each of them has 5 mismatch positions. Can anyone give me some suggestion? Can the partially complementary primers be used? How? Thanks Rating: Excellent! Reply

	By Seema on 16-Jan-2008 Hi, I am also trying to do this overlapping pcr but i am quite new to all this pcr stuff so can anybody give me a detailed protocol for the annealing part? including the pcr mix and cycling program?Thankns Rating: Excellent! Reply

By taqi on 12-Sep-2007 Cause i don't know your design, let's recapitulate: 1. You got a product A with Primer f1 and r1; 3' of the Amplicon is the overlapping part. 2. You got a product B with primer f2 and r2; 5' of the amplicon is overlapping. Right? Are the overlapping sequences really in the right direction??? For the hybridising pcr of the two amplicons take equal parts of product A and B as template in dilutions 1:10 - 1:100 - 1:1000 - 1:10000 (fading out primer r1 and f2. Primer in this reaction are f1 and r2. First step in the hybridising pcr is denaturation (as usual) at 95° C. And then: reduce to the Tm of the overlapping sequence (annealing and elongation of the two products, time depends of temp and length of the hybridised product). Followed by conventional PCR with 95° C, annealing/elongation and again. Still no product? Change hybridising temp onto both directions. Good luck and let me know the result. taqi Rating: Excellent! Reply

By Soumya Ghosh on 03-Sep-2007 I was trying to make a ligation between two genes by using the overlaping PCR method, but every time I get a PCR product which has a size one of the gene. This confirms that there is no ligation.My genes contain overalaping sequences at the the overhangs of the 3' and 5' ends of the gene 1 and gene 2 respectively. kindly suggest me so that I can proceed further. Rating: Excellent! Reply

	By smallhan on 06-Aug-2007 What's the suitable temperature of annealing? Rating: Excellent! Reply