Hello,
I am doing saturation mutagenesis at one position. The gene is cloned into a 9kb plasmid. By using reagents supplied by Quikchange XL SDM kit I set up a pre-PCR by using fwd and rev. primers sepeartely for 10 cycles. ((25ng template used and extension time was 9 min)). Then i mixed equal V of these two rxn, added Pfu again and continue to PCR for 18 cycle as said in the manual. Subsequently i did Dpn1 digestion for 3 hours ( 2 h more than kit suggets).. When i checked my product on gel there was a noise which i think could be digested template. My problem is the expected band was not there clearly, more like a smear and couldnt be differed from the noise.. (BTW i loaded 20% of the total PCR)
Is there any of you tried two-step PCR, wht should i expect to see on the gel, if only smear wht does it mean?
Thank you
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