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lalalala 04-06-2011 11:05 PM

PCR troubleshooting, Pfu Ultra II, whole worm DNA
Hey guys,

I'd appreciate any insights on how to troubleshoot my PCR.

I am trying to amplify a specific section of a gene inside the c elegans genome. This is what I've done so far:

1. DNA extraction -> yield ~2,200ng/ul

2. Taq PCR to check primers Tm. -> bright band, at right size (~1000bp) working fine for 53,54 and 55 Tms. NOTE: I did not dilute the DNA and used 1ul of original template.

3. Pfu Ultra II Fusion PCR, two Tms, 53 and 55. Diluted (100ng/ul) and not diluted templates. --> smears for samples with undiluted template, nothing for diluted samples.

4. Pfu Ultra II Fusion PCR, Tms: 49,50.1,51.1,52.1,53 undiluted template, small bright smear (near the bottom from 100 to 900bp).

5. Pfu Ultra II Fusion PCR, Tms: 48,49.2,50.2,52,52.9 diluted template (~100ng/ul) no bands at all, no smear, nothing.

Any suggestions? Is there something special I should be doing with the genomic DNA? Why do I see nothing with the diluted samples? I am thinking about trying higher Tms and/or play with the template concentration.... Please HELP!

jonoave 04-16-2011 01:50 AM

Re: PCR troubleshooting, Pfu Ultra II, whole worm DNA
For me, I'd stop messing around with the diluted and non-diluted templates and just stick to one. If you can get smears/bands with a particular template concentration using regular Taq, just stick with it for Pfu as well.

It sounds like you're getting some smears with undiluted template with Pfu, so I'd recommend sticking with that. Perhaps if you want, you can do a 5x or 10x dilution to save the amount of template used per reaction.

Some ideas:
1. You seem to be getting smears for Pfu up to 53 degrees. The first thing I'd recommend is do a temperature gradient and increase it gradually. See if there's a point between smears and no bands/smears at all.

2. Try using some enhancers, like DMS04 if you haven't, already. If you do use enhancers, you will need to increase the amount of Pfu or Taq you use.

3. Extension time - generally a longer extension time is recommended for Pfu, which is less efficient than regular Taq. Not sure if it'll help, but it's worth a try.

4. PCR master mix. Check the manufacturer's instructions and the volumes you're using for each solution. There might be differences between them.

E.g. - The reagents I use. For regular Taq the default options is 1.5 mM Mg, but for Pfu it's 2.0 mM Mg.

5. Keep your templates (and preferably, your reagents as well) in aliquots. Thawing and freezing repeatedly can cause damage and degradation of your DNA samples.

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