I have 6 primers for RAPD-PCR. Can I add all of the primers at the same time to the master mix? Or is it necessary to do PCR separately for each of the primers?
Dear Reza,
do the amplification reaction with individual primers. With unknown phage genomes, you will almost certainly find out that not all your primers will lead to a diagnostically interesting number of reproducible bands.
Good luck with your experiments.
Hi!
You might be aware of RAPD.
RAPD primers are decamers. You use just single primer per reaction, that work as both F & R. You get vast number of primers no idea what series you have with you. Some of the primers work very fine. You get more than 10 bands. Some may not you hardly get bands. Analysis takes time. Suppose if you mix all 6 primers they amplify good then it is difficult to analyze. I am attaching a picture for information. By seeing that imagine how it would be if you mix all primers at a time. Do it leisurely one by one.
Good Luck.
Very well Jetty, and dear Reza,
please take also into account that using primer mixes does not necessaily lead to additive band patterns of the results obtained with individual primers. One of the reasons is that there is competition between amplification products under the non-stringent RAPD conditions. By tendency, this effect leads to less bands.
Another reason is that you have much more binding sites now that lead to amplicons of appropriate length. This effect, by tendency, will produce additional bands, starting at one end with a different primer than on the other end. The more-than-one-primer approach can, in principle, be used for analytical purposes, but needs special optimization.
Cheer up - Johannes.
You might find the following paper useful; the full text can be downloaded off of ResearchGate: http://www.ncbi.nlm.nih.gov/pubmed/15081604
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