Improving PCR succes with low quantities of DNA

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hazelperry
hazelperry's picture
Improving PCR succes with low quantities of DNA

I am trying to amplify DNA from faecal pellets of the water vole.  I have tried several different extraction techniques and up until christmas was getting a faint band of product from the PCR's I was doing.  However since christmas everything has stopped working!  I've tried replacing all of my PCR reagents, extracting new samples, using both microsatellite primers and mitochondrial (the mitochondrial ones are the ones that worked before christmas.  I'm fairly certian its the PCR thats going wrong not the extractions becasue the PCR on samples that were extracted before christmas (and gave a product before christmas) now dont show product after the PCR.  I'm going to try chaing the quantities of BSA I'm using, increasing the number of cycles (although I'm already doing 35), increasing the time of the elongation step and adding DMSO and I cant seem to find much in the literature which is helpful. 

Does anyone have any other suggestions of things I could try to get it working?

DougB
DougB's picture
Hi Hazelberry,

Hi Hazelberry,

I realize this answer is long overdue. Unfortunately, I just joined. Hopefully you have found the solution to your problem already.

Nested PCR is one option. Of course this would depend on whether you can get a band from the first round of primers. If not, nested PCR probably does not work. Something we have had some success with is using either betaine or the Q-solution. You could also try changing the concentration of magnesium chloride or dNTPs in the PCR. Too much of either could inhibit amplification.

DougB