Posted 26 March 2011 - 02:35 AM
Iam trying to do invitro mutagenesis using quick change site directed mutagenesis kit from stratgene . I did not get any product after pcr using the manufacturers instruction.
5 μl of 10× reaction buffer
X μl (5–50 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
ddH2O to a final volume of 50 μl
1 μl of PfuTurbo DNA polymerase (2.5 U/μl)
1 95°C 30 seconds
95°C 30 seconds
55°C 1 minute
68° 5 min
Im not able to see any product in the gel and got no colonies after transformation after Dpn I digestion.
I dont knw if the annealing temperature should be 55 according to the protocol or according to the primers.
I have repeated the expt many times, without any success. Any help will be greatly appreciated
68°C 1 minute/kb of plasmid length*
Posted 26 March 2011 - 03:10 AM
do you used the primer design tool that is offered by stratagene (i think now its AB because they bought stratagene)?
how many kb is your vector?
you can try doing a gradient pcr if your annealing temp does not work and try to determine the best annealing temp for your PCR.
Posted 26 March 2011 - 07:10 AM
95/ 1 minute
then 18 cycles (perhaps a few more) of
95/ 30 s
55/ 30 s
68/ 1 min/Kb
Finally, a 5 minutes at 68
Posted 27 March 2011 - 12:08 AM
Posted 27 March 2011 - 01:04 AM
you have to verify that your template plasmid is really in supercoiled content to a large extent ...otherwise this will be also a source of trouble!