I use the same primer set and same conditions. The first PCR product from cDNA gave about 800 bp. Reamplification of the template gave 700 bp. A second amplification gave 600 bp.
Interestingly, I did an amplification of the original cDNA today and I got 600 bp. Any ideas whats happening? All this is happening within a week with products stored at -20 deg celsuis.
Thanks
The salt content and also the volume of sample and also the presence of dna dye in the gel or post staining can all have an effect on sample mobility in agarose. Possibly the salt content of the loaded sample has decreased so the sample runs quicker. You do not say what the correct size of the amplimer should be but it would be interesting to know.
Hi,
the problem might be very trivial: a variation in your electrophoresis conditions. TBE/TAE if not exchanged is getting more concentrated, same with agarose - it gets more dense because due to evaporation during repeated melting.
Try running all the PCR batches you have on the same gel. If there will be no difference, than there is no problem :)
If there will be difference...hmm... Do you get sharp bands or they are diffused?
Are you running the subsequent PCRs using amplicons as templates?
OR
Are you getting different results every time you run a PCR?
I think Anton Slyvka has a good idea. It would be interesting to see a sample of each amplification on the same gel
Vipul Batra amplicons of one round were used as template for the next round.
Anton Slyvka I usually run all 20 ul reaction product on the gel and I do not have remaining samples from previous reactions.
One thing I forgot to mention is that I used a different gel loading dye from what I normally use. Could this be the issue? I also use SYBR green gel stain and TAE.
Oh and the bands I get are sharp and distinct.
The salt content and also the volume of sample and also the presence of dna dye in the gel or post staining can all have an effect on sample mobility in agarose. Possibly the salt content of the loaded sample has decreased so the sample runs quicker. You do not say what the correct size of the amplimer should be but it would be interesting to know.
Paul Rutland desirable product of 800 bp, which was what I had after the very first cDNA PCR. The difference between the current and previous loading dye is that the previous one contained no SDS. I'll try the electrophoresis again using the 'no-SDS' dye and see if the band reads again at 800 bp.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology