I ran 35 PCR cycles on six gDNA samples using 18.5ul of a master mix (made up of goTag, two forward primer, two reverse primers, nuclease free H20) and 1.5ul of DNA. The primers are specific for genotyping this line of fish and have worked for the last 6 months. However, the gel with these samples, show three samples with the bands in the correct position (193bp, 295bp, & 434 bp) but the other three are double that size (~900bp, 530bp, & 430 bp). The primer dimer product all showing
Hi,
could you pls give a bit more details about the setup? PCR on gDNA/cDNA? Do your primers are specific or might there be isoforms? Singleplex or Multiplex etc? This will help because with these infos there is a lot of possible reasons but it does not help to speculate before having all infos which are needed
Sven
Hi,
thaks for the details. First of all it might be possible that the samples differ in purity. There are several small molecules whic mihgt be copurified which affect the PCR robustness. The mix has also not much Magnesium especially if you use more thna one primer pair. So I would on the one hand recommend to use a Multiplex Mastemrix and on the other hand a mix which is resistant for these small molecule inhibitors. If you are interested I can give you a suggestion as I am working on such products already for some time.
Beside this I wanted to ask if you have tested the PCR primer in singleplex and also ina gradient PCR? It might be possible that they are affecting each other in presence of non optimal DNA/ Presence of PCR inhibitors
Good Luck
Sven
Hi,
If the annealing temperature has been already standardized and made sure that no nonspecific amplifications occur,, then it could be some contaminating gDNA....Just a guess, but could it be some microbial contamination in the dead larvae? You can try to eliminate this possibility. Also for the dead larvae gDNA samples you can try to slightly raise the annealing temperature for more specificity and elimination of nonspecific amplicons.
Hi.
I do agree with other scientists opinions already given.
The one other aspect I would like to pinpoint is about your words "dead" and "deceased". In the case you're using it as I am thinking, may, it may be part of the trick.
Genomic DNA let stand for quite sometime in a dead larvae may be quite different of a "right now"deceased one. Get it? So, maybe, although they may seem quite equal based on DNA quantification and purity, they may be really different and the dead cells release of proteins may act on your reaction pattern. Ok?
Hope it helps.
You may check physical location of the primers, if the sizes are expected. Run PCR with primer combination f1r1, f2r2, f1r2, f2r1 in separate tubes and look for the result. You may also check temperature gradient pcr to optimize. PCR bands are never wrong, condition has to be optimized with proper reference control. Or the results has to be interpreted accordingly, if primer sets are universal or specific.
Thank you everyone. Great information. I think it comes down to the amount of time the larvae were dead prior to genotyping. I will keep this in mind and limit the amount of time between death and genotyping.
- Badges
- Science method