PCR on Leishmania DNA - (Jul/17/2005 )
i m working on Leishmania donovani's GP 63 protein. Its a surface protein and helps in virulence, basically help parasites to get entered in macrophages.
I am using primers for this protein. I tried a lot of combinations of different PCR parameters. But i did not get bands. The GC contents of primer is more than 60%.
Moreover , the DNA which is being used as template, is from subculture of indian isolate, which we are maintaing from last ten years.
So any body can let me know, can subculturing of such old isolate affect the Gp 63 gene in parasite genome? or its PCR problem.
Thanx in advance
hi! wat protocol u r using for PCR.
as for as subculturing is concerned, if there is no contamination it should nt effect the amplification. it is the problem with the concentrations u r using for PCR. recheck concentration of each component for PCr reax