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[HELP!!!] OE-PCR

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[HELP!!!] OE-PCR

Postby rainboo » Thu Jul 07, 2011 6:37 am

My PCR doesn't work.
I did OE first time, what can I do to optimize it???
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Postby JackBean » Thu Jul 07, 2011 10:37 am

what's OE?
http://www.biolib.cz/en/main/

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Postby canalon » Thu Jul 07, 2011 4:09 pm

Provide details of your problem.
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Postby rainboo » Thu Jul 07, 2011 4:33 pm

overlapping elongation
it uses the overlapping sequence of two or more genes to do PCR and ligate them without insertion at the same time
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Postby rainboo » Thu Jul 07, 2011 4:43 pm

I want to ligate two genes, one about 740bp and another 850bp.
I have had these two genes with a overlapping of 34bp and a pair of primers with Tm(60.3 & 69.0, these two primers are our projects' universal primers, though the Tm difference seems a little large, but they did well in general PCR)
I did OE-PCR for severl times these two days. At first, there is no target band, but with one almost at 750bp, I suspected it one of my templates. And then I did have a band, but it's only about 900bp. I don't know if the time of elongation is too short?(2min for Taq) or some other reason……
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Postby rainboo » Thu Jul 07, 2011 4:49 pm

And another problem!!!
Must the genes prepared for OE-PCR be replicated by enzymes with HIGH FIDELITY???
In my former lab, we always uses Pfu (and we needn't think about these problem)
However, here we only have Taq, Pfu Taq and Phusion, I'm not sure if I have to use Phusion or maybe there is some other ways to solve this problem for I have P these two genes using Taq……
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Postby rainboo » Thu Jul 07, 2011 5:52 pm

I'm sorry to find that maybe more people call it FUSION PCR
I want to know if anybody has its protocol or experimence…… help~~thx so much
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Postby JackBean » Mon Aug 01, 2011 12:49 pm

yeah, fusion PCR is good, even overlapping elongation if fine, actually anything else than abbreviation is great ;)

high fidelity enzyme is better, because it is suited to amplify long pieces of DNA, what's probably what are you targeting for ;)
If your amplicon should be around 1500 bp, 2 minutes should be fine
http://www.biolib.cz/en/main/

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