i am going to do RAPD analysis of two different plant species with 31 different RAPD primers. I need to know the best suitable concentration and volume of primer, MgCl2, Taq pol, PCR buffer, and each dNTPs for getting superb band pattern and enough darkness of bands on gel.
also need to know the ideal templet DNA concentration for good reaction.
HI, Jigar bhai
try this protocol which will give you best amplification of RAPD primers
50-75 ng of DNA is more than enough per a reaction. Annealing temp 35C work good. Pravin mentioned you can carry on 20uL. I used to do 10-15 uL.
What plant samples are you working with! Generally plant DNA contains many secondary products that may hinder the reactions some times. RAPD are very sensitive reactions, all primers may not work some times only single or two bands. Take many primers set reaction, out of them which primer gave good number of bands go with those primers.
Good Luck!!
Hi Jigar,
as RAPD primers can be quite different, the according optimal pcr conditions may vary quit a bit. We did quite a lot of work with HAT-RAPD PCR (in several species) using primers with high GC content and ISSR-like features. That allows for high annealing temperatures and very good reproducability. Conditions are for instance described in the papers linked here. If you cannot access the papers, please drop me a message and I send you the protocol.
Best wishes and good luck,
Klaus
http://journals.cambridge.org/action/displayAbstract?fromPage=online&aid=191797&fileId=S0021859603003447
http://link.springer.com/article/10.1007/s10722-006-9169-2
http://link.springer.com/article/10.1007/s00606-011-0570-8
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