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invitro mutagenesis using stragene kit

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#1 jaya2020



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Posted 30 March 2011 - 04:21 AM


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 l of PfuUltra HF DNA polymerase (2.5 U/l)

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

68C 1 minute/kb of plasmid length, 18 cycles
68C 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.



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