PCR amplification related..

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PCR amplification related..

Postby molbio2631 » Jun 11 2017 4:28 am

Hi all :)

Could u guys suggest me a good taq polymerase for amplifying a 5Kb, GC-rich cDNA.... I have already tried qiagen HotStart Polymerase and Thermo Phusion II Polymerases --- they gave positive results with my beta-actin but were unsuccessful with my template of interest.

Please help :cry: !
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Joined: Jun 07 2017 12:44 am

Re: PCR amplification related..

Postby ck24 » Jul 31 2017 11:44 pm

Kapa HiFi Biosystems (the one where the GC-Buffer comes as part of the PCR kit) should work just fine. Just be sure to carefully read the manufacturer's instructions. You could also try and do a 2 step PCR with Phusion Polymerase with GC buffer. Using a two step would be easier than trying to optimize the extension time of the last step of the pcr cycle and GC buffer will resolve any RNA secondary structures that could be forming during the reaction.
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Joined: Jul 31 2017 10:58 pm

Re: PCR amplification related..

Postby 29yrsExperience » Aug 11 2017 2:09 pm

If you are still working on this, a few suggestions. I assume you have already tried manipulating the Mg concentration, times, and temperatures, and added denaturants like DMSO or betaine. Do you get non-specific stuff or nothing? To find out whether you forward and reverse primers are binding and extending at all, try designing some new primers within the target that, when paired with your existing F and R primers, will allow you amplify a small segment (~0.5 kb) at either end of your full-size target. If those smaller targets won’t amplify, try redesigning your original primers to tolerate higher annealing temperatures, and try doing a 2 step PCR (both annealing and extension at 72 degrees) ‘till you get something that works on the small target. Then try the full size. If you have primers that work on the small targets, but you cannot force them to go the full length, you might have to divide your target into two overlapping regions and amplify them separately. Try to find a convenient unique restriction site in the middle, include it in both pieces, then ligate the two products at that site later. I’ve had to do up to 3 separate overlapping pieces.

As for the kit, use a proofreading long-range PCR kit- usually these have proprietary enzyme blends (not Taq) and optional buffers for high GC targets. I’ve used three brands and each one has managed to amp something the other two failed at over the years. I like one that tolerates denaturation temperatures of 98 degrees C. Good luck!
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