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[jaya2020]

Hi

Iam having problem in invitro mutagenesis. I am using kit from stragene. I did the cycle according to the maufacturers instruction. But nothing is working, even after repetitions. I designed the primers in their website. I didnt note the TM of the primers while designing. The primer of the Tm is around 68 deg according to the data sheet. The conditions used are

5 μl of 10× reaction buffer
X μl (10 ng) of dsDNA template
X μl (125 ng) of oligonucleotide primer #1
X μl (125 ng) of oligonucleotide primer #2
1 μl of dNTP mix
3 μl of QuikSolution
ddH2O to a final volume of 50 μl

1 ìl of PfuUltra HF DNA polymerase (2.5 U/ìl)




95°C 1 minute
95°C 50 seconds
60°C 50 seconds

68°C 1 minute/kb of plasmid length, 18 cycles
68°C 7 minutes


Will it work if I set the TM for the primer according to the data sheet, or should I go with the TM given by the manufacturer.

Any help will be greatly appreciated.


Regards,

jaya