let me share my frustration
i would like to make a site-directed mutagenesis on a vector but it has been failed again and again.
I mean I have never got the amplified mutant plasmid.
here is my protocol:
- x ul plasmid template (50 ng)
- 1 ul FW primer (10 pmol/ul)
- 1 ul REV primer (10 pmol/ul)
fully complementer to each other (36 bp , Tm: 83 C)
- 5 ul buffer (10X) [buffer contains Mg2+]
- 1 ul Pfu ultra II polymerase
- 1,5 ul dNTP mix (10 mM)
- x ul H2O
Vt: 50 ul
- - -
denaturing temperature: 95 C
annealing temperature: 55 C
extension temperature: 72 C
I don't know the "hidden factor" that wasn't to be considered...