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Amplifying environmental DNA extract with proofreading enzyme


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#1 petemanpeter

petemanpeter

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Posted 13 November 2009 - 11:57 AM

I need help getting Phu to work on environmental extracts from hot spring sediment samples. The DNA was extracted using the FastDNA spin kit for soil made by MP Bio. I have had some spotty success with getting Phu to work on other genes in the same extracts, but it still doesn't work every time even when I have had positive results using Taq. The gene I am trying to amplify has a high AT content and is around 200bp. The primers are degenerate and have a low annealing temp of 47C. On my control DNA I have to use the GC buffer in order to get it to amplify with Phu even with the high AT content.

I have also had better succes with getting Pfx to amplify genes from the extracts, however I am unable to get it to amplify my gene of interest even with control DNA.

I am looking for advice for how I may get Pfu to work on my evironmental extracts or getting Pfx to work at all. I'm really tired of messing around with this and I want my research to move forward! Help!!

~peter




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