1 reply to this topic

[Kristin H.]

Hi everyone,

I want to do PCRs for Illumina-sequencing (V4 region of eucaryotic 18s rRNA gene) but I always have bands in the negative controls (Master mix without template). I think I tried everything: milli q water, several sterile filtered water, also bought water for real time q-PCR, new buffer, new dNTPs, new polymerase, new pipet tips, new tubes, I did it at two different clean benches in different rooms, also at my normal working place (no clean bench), a collegue did the same and she has also a band. The bands are always there and have always the same extent.

So I wonder if the Primers are contaminated. Thus I did a PCR for 454-sequencing to test the components of my Illumina-PCR. The 454 PCR have other components as for the illumina-sequencing and also different primers (for the V4 region of 18s rRNA genes, as well), which are shorter than the Illumina-ones. The result: the negative control was as it should be. however, where I used a primer as template there was a band at the same height as the positve control.

Can I use Primer as template or will they amplify eachother? I know that if there are primer dimer, they have a shorter length than the expected product. But that is not the case.

Can someone help?

[Wunder]

Hello

Hmm well I suppose in theory there is a small chance that one primer could anneal to the very end of the other primer and use it like a template. But it will only ever be as large as your two primers over lapping so if you're getting a really large band in your negative control this can't be the reason (also if they are annealing then it technically is primer dimer). If this is the reason you could try higher annealing temperatures.

So let me get this straight. You tried new primers (454) on your negative control and got no bands but when you tried using your Illumina primers as a template and your 454 as the primers you got a band? Are you sure your Illumia primer stock isn't contaminated with DNA?