double digestion time - (Nov/19/2008 )
i have to do double digestion of PCR products with BamHI and KpnI (fermentas).
they recommend bam Hi buffer for both with 2 fold excess of kpnI.
incubation is at 37 degree but time is what i dont know??/ can any one help.
also how to proceed- both enzs simultaneously or sequentially?
one more thing- for doing 3 piece ligation does dephophorylation required/recommended.
thanks a lot
-beulah-
QUOTE (beulah @ Nov 19 2008, 10:25 PM)
i have to do double digestion of PCR products with BamHI and KpnI (fermentas).
they recommend bam Hi buffer for both with 2 fold excess of kpnI.
incubation is at 37 degree but time is what i dont know??/ can any one help.
also how to proceed- both enzs simultaneously or sequentially?
one more thing- for doing 3 piece ligation does dephophorylation required/recommended.
thanks a lot
they recommend bam Hi buffer for both with 2 fold excess of kpnI.
incubation is at 37 degree but time is what i dont know??/ can any one help.
also how to proceed- both enzs simultaneously or sequentially?
one more thing- for doing 3 piece ligation does dephophorylation required/recommended.
thanks a lot
there's no say on this issue. some like to do overnight some likes to go for 1-2 hours . so on so forth.
i believe for ur case, 3 hours would be ok.
-Hanming86-