I am running PCR on my samples, and I've been getting intense amplification from negative control samples. I've ran 3 samples, (1 is target, 2 is NC and 3 is NC without primers) for PCR step. From both target and NC sample, I've got intense amplification and from the gel electrophoresis, the sizes are similar too, although the NC sample is slightly smaller in size like 50bp or so. However, I did not get any amplification from NC without primers, so I am wondering if I am getting nonspecific amplification of my primers to the NC gene. I've already tested annealing temperature.
Yes... primer dimer enticed product could be giving you your 'non-specific' signal here... what are the primer dimer possibilities in your primers? Paste the sequences of your primers into an available primer analyzing program (Primer Express, Primer 3, NCBI tools, etc.) and check if you have that pesky possibility afoot...
It might be because of contamination also, or you can check specificity of your primers.
Hi Han,
Always check your primers for any interaction using the insilico tools mentioned above by Jack. This can give you prior indication of possible problems in the PCR assay... These can also be primer dimer's..try to keep the amount of primers minimum possible for clean amplification of the deired product....
bye
Yes, there might be possibility of primer-dimer formation! For this, please check the primer-dimer formation chances on available primer analyzing program like NCBI tools, Primer express etc.. These programs tell you about this occurrence rate in your primers. You could also check contamination on your reagents also by using the primers and probes from multiple lots (i.e. using new unused vials of primers and probes if possible)...
- Badges
- Science method