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Topic Started by parvoman
on 2/22/2009 16:34 PM
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I'm about to do a PCR cloning in which a 3.8Kb fragment is to be cloned using Clontech's In-Fusion kit. In the past I have used NEB Deep Vent and more recently Pfusion. Of those of you who do PCR cloning and need the efficient proof-reading function, which pol do you use? Thanks
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Posted By Ivan
on 2/22/2009 8:53 AM
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This may be a little outdated, but a few years ago a colleague of mine told me that she got best results using a mixture of two enzymes (1:1): AccuTaq (from Sigma) and Pfu (Fermentas). If this did not work, you can always spike some regular Taq (to get more amplicon). I assume that there are new proofreading enzymes out there that are even better, yet the idea I think still holds: if unsure of which enzyme is best and if it possible, try a mixture of enzymes. That way you may be able to harness the best of both worlds, assuming that each enzyme has an advantage over the other because of different reasons.
Ivan Delgado Orlic Carlsbad, CA
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Posted By RLS
on 2/22/2009 11:27 AM
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I typically use Stratagene's PfuUltra II Fusion HS DNA polymerase. It's pretty fast, and it has great accuracy.
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Posted By parvoman
on 2/23/2009 15:40 PM
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| Ivan said: |
This may be a little outdated, but a few years ago a colleague of mine told me that she got best results using a mixture of two enzymes (1:1): AccuTaq (from Sigma) and Pfu (Fermentas). If this did not work, you can always spike some regular Taq (to get more amplicon). I assume that there are new proofreading enzymes out there that are even better, yet the idea I think still holds: if unsure of which enzyme is best and if it possible, try a mixture of enzymes. That way you may be able to harness the best of both worlds, assuming that each enzyme has an advantage over the other because of different reasons.
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I don't need to have extra sensitivity because I'm using a plasmid as template. Does anyone else have a recommendation?
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| parvoman said: |
I don't need to have extra sensitivity because I'm using a plasmid as template. Does anyone else have a recommendation?
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In that case, I would just use Pfu or Pfu Turbo from Stratagene (or any vendor, really). Unless you're trying to amplify something very long, you should get plenty of amplification with 30-35 cycles of PCR using Pfu. Use an extension time of 1 minute/kb. I only mix Pfu with Taq if I have a long target from genomic DNA or cDNA.
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