I'm having problems amplifying the intergenic region (IGR) of the ambisense Tospovirus (IYSV). This region is AT rich and has loops. For this I've tried different primer pairs and tried optimization using all possible methods.
Vishal Gurav
Thanks Amanda and Himashu for ur kind reply, I'll definitely try to follow ur suggestions.
but Earlier i've used different specific primers with various annealing temperatures. I've also used Pfu DNA polymerase but positive results r not yet found.
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Lieven Gevaert
better use a pfu taq mix and or hot start pfu
http://www.gentaurpub.com/Tospovirus/VG9zcG92aXJ1cw==.html
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Vishal Gurav
Thank u all,
Especially Amanda, your suggestion really worked. Thanks a lot.
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