I have a prolem with my PCR products. For the fist time I used PCR then tooke the products on gel electrophoresis, with the published protocol, my results became positive. For the next time the same PCR products with same protocols and samples were negative on gel electrophoresis. I am a M.Sc student and don't know what should I do. If there is someone who had the same problesms it would be great to help me.
Thank you
Hello,
It's quite possible that PCR products have undergone degradation. For the next time, make sure the reaction is stopped and products are well conserved at -20 or -80 degrees.
Good luck
Hi
Its happened with me few times at the beginning got result and in the next time nothing. My advice just repeat again with the same steps that u did before (don't change anything in the protocol). Concentrate more and work fast to avoid misprim. If you are working slow, try to use hotstart PCR reagent. Keep in your mind to reserve your PCR product, DNA samples and PCR reagents within -20 or -80 C. To avoid DNA degradation divide your DNA samples into two parts: small part, keep it in -20 C for your usual test and other large part, in -80 C to keep longer time. (:
There are several reasons why this could happen, but first, are the size of the products you detected matching the predicted amplicon size? Did you do a no template control and was there amplification in that? Are you amplifying from cDNA that you've synthesized or Genomic/plasmid DNA? If you made cDNA from RNA, how is the purity and quality of your RNA (260/280 ratio and 28S and 18S RNA ratios)? Sometimes nucleic acid extractions contain impurities that can inhibit the RT and PCR reactions so you can purify the DNA/RNA again by phenol chloroform and check the 260/280 curves for contaminating protein or phenol/ Guanidine thiocyanate (in Trizol extraction reagents).
Like I mentioned there are so many reasons why this could be happening, but the points I mentioned are good places to start troubleshooting.
you have a good starting point. The first pcr worked. It is important when running pcr to have both a negative (no dna sample) and positive ( a sample known to amplify ) control in every experiment. run one of these samples in your next pcr. Then if there is no amplification in all samples you can be sure that there is a problem with the pcr and not just that your samples are negative. for the amplicon
I would make up new reagents particularly the primers ( which should be dissolved in TE ) and run the pcr with annealing temperature 2c lower and with more magnesium and 3-5 more cycles of pcr The results may be a bit dirty but it is easier to clean up a working pcr with too many bands than it is to work out why there is no amplification as this can be caused by very many factors. You need to run a size standard on your agarose gel as sometimes you get a very small product which is primer dimer where the primers stick together and this can sometimes looklike a diffuse product but actually primer dimer stops the correct pcr from working.
Dear researchers, my friends, I think with your recommendation my work will OK and I am thankful for that.
Dear Alireza,
It happens. Usually if you store PCR product in -20 or -80, the chance of getting different result is high. If I am in your position, I will finish both gel electrophoresis at the same day either in two electrophoresis tool or one after another. Or use in one electrophoresis twice.
Good luck Alireza
at last, after lowering annealing tempreature by 2 degrees and running gel electrophoresis at the same day of PCR, I get the results. And this is because of your helps, Thank you verymuch
Dear Alireza,
Thank you for let us know you accomplished the task.
Good luck for next steps
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