I have been struggling to identify the source of my contamination in my RT -ves. My primers are designed around large introns such that genomic DNA contamination if little should not amplify. My water is always clean but my RT ves control show the same melting curve as my experimental samples, and when run on a gel correpond to my cDNA amplicon size. I have changed mastermix, plates, seals, etc. to no avail! Since my water is clean, I presume my reagents are OK. I cant seem to think of what to tweak to get rid of this contamination. Please help!!!!
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ivanbio
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Posted 02 March 2011 - 10:22 AM
Is it possible that your amplicon is a primer dimer? I assume that is not the case, but if you have an amplicon that is ~100 bp in size then maybe that is the problem.
Alternatively your primers may be amplifying a different DNA region other than the one you think you are amplifying, which happens to be the same size as your desired amplicon. As easy way to get around that issue would be to simply design a new set of primers.
A good control when this kind of issues arises is to have a second assay you know works very well, and does not amplify background/contamination signals, and run it side by side. If you get an amplicon in your RT -ve with this control assay too then you know the issue is with your reagents.
Cheers
Alternatively your primers may be amplifying a different DNA region other than the one you think you are amplifying, which happens to be the same size as your desired amplicon. As easy way to get around that issue would be to simply design a new set of primers.
A good control when this kind of issues arises is to have a second assay you know works very well, and does not amplify background/contamination signals, and run it side by side. If you get an amplicon in your RT -ve with this control assay too then you know the issue is with your reagents.
Cheers
Ivan
Carlsbad, CA
