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-   -   Sequencing whole cDNA using PCR (http://www.molecularstation.com/forum/pcr-polymerase-chain-reaction-forum/87765-sequencing-whole-cdna-using-pcr.html)

Sialic Acid 04-03-2013 03:06 AM

Sequencing whole cDNA using PCR
 
Since I'm a bit of a novice at molecular biology, I thought I would ask before spending a lot of money on kits and reagents in order to try to sequence whole cDNA using PCR to clone it.

The 2 of the cDNAs I'm after are approx~1.5 kb long. The third one is ~5kb long. I've looked into some high high fidelity pcr kits, and the protocols say that they should be able to amplify >5 kb amplicons no problem.

The problem I have is, how should I design a primer for such a long pcr reaction? I'll start running into primer dimers and hairpins since the reaction is much longer right? The sequences of the whole cDNAs are known (I'm looking for mutations), and I designed my primers to work at the very beginning of the 5' and 3' ends. The problem is that it is virtually impossible to create primers that don't give self complimentary scores <3.0 according to primer blast. Any tips or can I just use such primers anyway? I'm under the impression that primers don't have to be perfect for pcr to work, correct?


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