2 replies to this topic

[fishgirl]

Hi,

I am running isolated fish DNA with CR-A/CR-E primers and I am having some of the samples not working, I have checked DNA concentrations and all are above 25ng/ul which should be ample and not over saturate either. I am using 25mM of Mg, not sure how to continue with the trouble shooting? try diluting the DNA? any tips...lower Mg? raise Mg higher??

[bob1]

25 mM Mg²⁺ is a lot, the normal range is 0.5-5 mM... did you optimise these primers for this PCR?

If not, try titrating the Mg²⁺ to see what works best, and try an annealing temperature gradient.  

If the PCR works for most samples, but only a few are not working, those ones may have inhibitors in them, try diluting the DNA out 1:100 or re-precipitate it and resuspend in 15 mM tris Cl pH 7.5.

[fishgirl]

Thank you, sorry I meant 3mM of MgCL2, per tube

I will try diluting the DNA, yes 90% of the samples ran fine so must a problem with the extractions, the inhibitors would make sense