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Problem with Genomic DNA PCR

Problem with Genomic DNA PCR - PCR - Polymerase Chain Reaction Forum

Problem with Genomic DNA PCR - PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.


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Old 09-10-2010, 06:45 AM
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Default Problem with Genomic DNA PCR



Hi, I am trying to PCR 1-2kb target regions from mouse cell genomic DNA.

I have tried using GreenTaq from Qiagen and it works, but when i change to the high fidelity KOD enzyme, the PCR reaction does not work.

My conditions for GreenTaq are:
25ng of gDNA per 25ul reaction
5% DMSO
annealing temp 55 degrees , 30s
elongation temp 72 degrees , 1 min 30s
28 cycles

I have basically carried out the same conditions to KOD, but it has not worked so far.
Subsequently, I tried varying the amount of gDNA from 7 to 100ng, increasing KOD enzyme from 1U to 2U, increasing the [Mg2+] from 2.5mM to 3mM, as well as decreasing the annealing temperature to 50 degrees.

Any suggestions on what could be the problem, and what I could do now?
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Old 09-10-2010, 07:54 AM
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Default Re: Problem with Genomic DNA PCR

There may be an inhibitor of a PCR reaction in your genomic. Try diluting it in various amounts to optimize.

Also make sure you vary your Magnesium and your DMSO. DMSO from 1-5%. 5% is on the high end, but it worked for me on a 80%+ GC content sequence.

Genomic DNA needs to be pipetted many times to get rid of the secondary structure, and carefully optimized for amount of DNA and +/- DMSO.

They are the tougher PCR's

Post your gels here and I can take a lot (each lane should have a different condition to optimize).

cheers
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Old 09-20-2010, 03:42 PM
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Default Re: Problem with Genomic DNA PCR

Just some comments. Which KOD version are you using? I'm using KOD hotstart and the recommended extension time for 1-2kb is 20s/kb. 90 sec is too long and may lead to no product according to the KOD hotstart user manual. Optimum extension temperature with KOD hotstart is 70C. What's your denaturation temperature? Also, the exonuclease activity of KOD may make a difference in terms of sample prep and primer design (longer primers). You may need to read the user manual and start with the recommended parameters instead of using those for GreenTaq on KOD.
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Old 09-22-2010, 01:24 AM
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Default Re: Problem with Genomic DNA PCR

Hi,

Thanks a lot for the help. Yes, I'm using KOD hotstart enzyme. Previously, I tried tinkering with the MgSO4 and KOD enzyme levels independently, and I couldn't get any bands at all (despite getting clear bands using the Qiagen Green Taq enzyme).

I think the breakthrough came when I essentially doubled all the volumes by doing the same reaction in half the volume (by a freak accident). There was a gradient of smears (a happy thing as something slightly wierd is always better than nothing at all), and the smear intensity was proportional to the amount of template DNA (thx to suggestion on varying template DNA concentration! ). I realised that amongst the intense smears, there were signs of bands coming through.

I subsequently did optimisation at this template DNA concentration, which was surprisingly high at about 400ng. I tinkered with the KOD enzyme conc and MgSO4 conc (sound advice too!), roughly doubling them, and started getting clearer bands.

I still don't quite understand the mechanics of all this though, since some insert PCRs worked at essentially 25-50ng template, while PCR of these other genes required 400-500ng of template DNA, with no obvious sign of template inhibition. Of course, I have since tried out a third set of genes, which are inhibited at 400ng of template DNA and require about 70ng template optimally. I guess it might be due to the accessibility of the gene to primer amplification, although any further suggestions/explanations are highly welcome.
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