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PCR amplification with Pfu / quality of DNA


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#1 PiDC

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Posted 18 November 2010 - 06:51 AM

Hi:

We are having problems with PCR using the enzyme Pfu (ultra or turbo). I need to use this enzyme instead of Taq pol becuase I want to sequence mutations in the PCR product so I need the highest fidelity enzyme.

I do not get any PCR band when using genomic DNA. I have changed the stocks for everything (dNTPs, primers, buffers, enzyme, template...), and I get the band only when using a plasmid instead of genomic DNA as the template for the PCR.

I purify the genomic DNA with the standard protocol: lysis with proteinase K overnigh, phenol extraction and ethanol precipitation. The A260/280 of the DNA I obtain is above 1.8, but maybe the its quality/purity is not enough for this PCR.

Does anybody know how to improve the quality/purity of the genomic DNA during the purification?

Thank you very much!!

#2 phage434

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Posted 18 November 2010 - 12:49 PM

How much genomic DNA are you adding? Too much DNA can cause failure of PCR. Make sure you have little or no alcohol in the DNA. You could try ethanol precipitating it and making sure that the pellet is alcohol free before resuspension (smell it).

#3 christy

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Posted 18 November 2010 - 05:25 PM

Hi:

We are having problems with PCR using the enzyme Pfu (ultra or turbo). I need to use this enzyme instead of Taq pol becuase I want to sequence mutations in the PCR product so I need the highest fidelity enzyme.

I do not get any PCR band when using genomic DNA. I have changed the stocks for everything (dNTPs, primers, buffers, enzyme, template...), and I get the band only when using a plasmid instead of genomic DNA as the template for the PCR.

I purify the genomic DNA with the standard protocol: lysis with proteinase K overnigh, phenol extraction and ethanol precipitation. The A260/280 of the DNA I obtain is above 1.8, but maybe the its quality/purity is not enough for this PCR.

Does anybody know how to improve the quality/purity of the genomic DNA during the purification?

Thank you very much!!



#4 christy

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Posted 18 November 2010 - 05:32 PM

Try pfx 50 polymerase from invitrogen. For GC rich dna u can use GC rich dha polymerase.

In korea, we are using one local brand "solgent" that is band doctor, u can use along with pfx polymerase, it gives us very good result. even for GC rich dna.

Good luck !!!!!!!!!!!!!!!!!!!

#5 philman

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Posted 23 November 2010 - 07:44 AM

When I started using Pfu I had a similar problem, my problem turned out to be MgCL2 concentration, try doing a titration of different Mg concentrations with your PCR, I got best results for a 2kb length at a 4mM concentration of MgCL2.




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