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Sequencing whole cDNA using PCR

Sequencing whole cDNA using PCR - PCR - Polymerase Chain Reaction Forum

Sequencing whole cDNA using PCR - PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.


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Old 04-03-2013, 03:06 AM
Pipette Filler
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Default 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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