I want to amplify the whole nrITS region with universal primers ITS4/5 (White et al.,1990). Can anyone suggest any PCR optimizations (PCR mix and cycle) to amplify it successfully?
Hi Biswajit
In my opinion you can use conditions exactly used in the paper 'White etal., 1990...If you find some problem then go for optimizations..
bye
Dear, Biswajit
You have find this conditions in White et al., 1990. But in many cases you need to make changes especially in annealing temperature and sometimes in denaturating temperature. These conditions are highly dependent on the material and the object of research.
When I used the same primers in my work, I had to reduce the annealing temperature to obtain good results.
If your DNA contain many GC pairs sometimes necessary to use a higher denaturing temperature. Also a lot depends on type PCR you are conducting - PCR with previously isolated DNA or direct PCR.
If you give me some information about organism you study, I wil try to help you.
Best wishes
when DNA is GC rich, you can use DMSO to reduce annealing temperature.
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