5 replies to this topic

[jaya2020]

hi ,

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
Then add
1 μl of PfuTurbo DNA polymerase (2.5 U/μl)


1 95°C 30 seconds
95°C 30 seconds
55°C 1 minute

18 cycles

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

thanks,

jaya
68°C 1 minute/kb of plasmid length*

[pDNA]

isn't the annealing temp around 60°C using the quickchange kit?

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.

Regards,
p

[Ameya P]

Increase number of cycles... I would say.

[phage434]

Are you doing an extension at all?  Your cycle listing could be interpreted as cycling between denaturing and annealing, with no extension.  Your cycle should be:

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

[jaya2020]

Thank you all for the reply. I will try to do as suggested.

regards,

jaya

[pDNA]

another thing that came to my mind:
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!

Regards,
p

jaya2020, on 27 March 2011 - 12:08 AM, said:

Thank you all for the reply. I will try to do as suggested.

regards,

jaya