I am having trouble with designing a primer for cloning Y2H.
My full length RNA sequence from source lacks 5'-UTR region, it starts at ATG, then I must design a primer from ATG. The trouble is high GC forward ATG, then my primers can not work well in case 20 mers. I reduced to 15 mers, I got a band but not the one I need.
Can anyone can help me this case? I use pAD-GAL4-2.1 and pBD-GAL4 Cam vector for cloning.
Thank in advance.
You can try another DNA polymerase that can get through GC rich regions or try altering your PCR conditions by the addition of DMSO for example.
You can add 10% DMSO or glicerol for better results, additionally you can use high denaturing temperatures or longer times for initial denatiralization (that's what some people recommended to you use a hot start polymerase such as TaKaRa). Finally you can try use lower template and primers concentrations.
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