I have been getting a band in the second (nested) amplification of the negative from the initial PCR, but not in my negative control testing for contamination. Any ideas?
Information:
I am using a nested PCR for different Ehrlichia species in whole blood.
The initial PCR is an Ehrlichia screen using one set of primers. For 20 cycles.
The nested PCR's have specific primers based on the Ehrlichia species. For 30 cycles.
The initial negative controls have RNases free water, Taq polymerase, and the Ehrlichia screen primers.
When I run the nested, I take 1 uL from the initial negative (which is clean on the gel).
I've recently changed out my reagents, and the band is around 500 bp so not primer dimers.
Could be something specific in the initial primers but I only see it in the samples that should be negative and it's a pretty big piece to be related to the primers.
Thank you
50 rounds of pcr is a huge amplification. To minimise primer interactions and to minimise weak false bands amplifying it is worth running 1ul of 1:100 and 1:1000 first round product which should be plenty to give a strong band. It means also that you would dilute your NTC sample 1:1000 or 1:100 to be a proper control for your template pcr and the dilution may get rid of the very slight contamination that the NTC is showing. Due to the nature of pcr 10 cycles of pcr is 1000 times more amplimer so mathematically ( but not in real life) 30 cycles of pcr could be 1,000,000,000 times more amplification so even the tiniest contamination will be picked up with a 30 cycle pcr and between the 1st and 2nd pcrs you are running 50 cycles.
I thought I'd give an update:
Most of the time, diluting the initial PCR negative did remove the band in the nested product. However, it was random, and sometimes it was still present, although faint.
Next, diluting the PCR products would sometimes cause the bands to be very faint or not be visible.
So it could work for someone else who is having the same issue.
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