Hello researchers
i'm trying to perform a RAPD genotyping for wheat samples . I need any ideas which help me how to start and proceed in optimization of this reaction , and what are the critical variables ?
i have gotaq green mastermix from promega and a set of decamers primers .
thank in advance for any help .
You could use the following paper
https://www.researchgate.net/publication/274082895_Genetic_Polymorphism_among_Some_Sugarcane_Germplasm_Collections_as_revealed_by_RAPD_and_ISSR_analyses
Article Genetic Polymorphism among Some Sugarcane Germplasm Collecti...
Khaled A M Khaled : this paper say that you use 15 ng of the primers . is this normal or typing mistake ?
I don't think you find many problems if you use a PCR mastermix and just test the 10-mer primers you want to use. From that standpoint, DNA quality is your only variable and should be the one you have to take care the most. If you use a commercial kit like Qiagen Dneasy you should be fine but common extraction methods like CTAP should also suffice (and is cheaper). I think 15 ng of DNA is in the range of what you need, we used 17 ng for potatoes. What you do is to dilute your DNA to a 10 ng/uL concentration so then you just add 1.5 uL to the mix to have the 15 ng.
Alfonso. H. Del Rio, thank a lot : and what about primers concentration . what are your recommendation about primers starting concentration .
We don't do RAPDs anymore but as far as I remember a primer working concentration of 10 mM should be OK, We used to put about 1 to 1.5 uL of this to the PCR mix.
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