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Can I use Primer as template?

PCR Primer illumina contamination

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#1 Kristin H.

Kristin H.

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Posted 24 July 2013 - 04:43 AM

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? :)





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