QuickChnage Mutagenesis problem - no mutation, please help!!!!!!!! (Jan/02/2008 )

Hi all, i've 3 week trying to do a site direct mutagenesis, but anything works, my plasmid is pEYFP-N1, my insert is 2.3 Kb. I am using the Stratagene's kit QuickChange Lightning, and after the transformation, the only thing that i have is the parental plasmid, no mutation at all... I am really desperate, the kit is really expensive and othing works...

My procedure is:

5ul of 10X reaction buffer
3ul of Forward primer (125 ng)
3ul of Rverse primer (125 ng)
1ul of dNTP mix
1.5ul of QuickSolution reagent
36.5ul of H2O
Then i add 1 ul of QuickChange Lightning enzyme.

The cycling parameters for 18 cycles are:

95 C 2 minutes
95 C 20 seconds
68 C 10 seconds
68 C 3.2 minutes

after that i add 2 ul of DpnI and and incubate at 37 C for 5 minutes.... I increase this time and the enzyme for almost 2 hrs and no change at all...

I transform the reaction, and a lot of colonies but with no mutation.
The other thing that i did, was to purify the PCR reaction and concentrate it in 10 ul, and then i digested again with a DpI from NEB, and then i transform it, but nothing works...

Please if someone can help me !!!!!!!!!!!!, i do not what else can i do.....

I guess you must have already checked the primer sequences. I would get the program to redesign it and order it again even if its the same.

i say to order even if its the same because, sometimes, primers do end up with a wrong base, this is rare but it can happen. It happened to us.

Dpn 1, digestion, do it for 1 hr.

All of the reagents for mutagenesis can be bought separately and this reduces the cost considerably compared to buying the kit. Actually, you only need the enzyme and everything else is usually in the lab.

Hi, thanks for the advise, i'll send the again. i have another questions, have you tried the AccuPrimePfx of invitrogen, to do site direct mutagenesis?...

The cycling parameters for 18 cycles are:

95 C 2 minutes
95 C 20 seconds
68 C 10 seconds
68 C 3.2 minutes

after that i add 2 ul of DpnI and and incubate at 37 C for 5 minutes.... I increase this time and the enzyme for almost 2 hrs and no change at all...

I'm sure you don't mean you perform the primer annealing step at 68oC?? Was just a typing mistake, huh?
Anyway, what we do is we always perform the primer annealing step at 55oC and then digest the mixture by adding 1 ul of DpnI and incubating it for 2h at 37oC. And then we repeat the digesting step once more. Some of my colleagues even perform the digestion reaction O.N. However, we are are using the "old" (i.e. non-lightning :-)) QuikChange kit.
We usually decrease the reaction volume to 25 ul - so we can perform twice the number of reactions with one kit. Works just fine.
Just another couple of thoughts:
- I don't know the amount of template DNA you are using, but I would recommend not using more than 20 ng
- Check the primer concentrations (NanoDrop or similar)
- Try using new solution of dNTPs (seemed to be a problem at some point in our lab!)
- Make sure the DpnI works (use on any E. coli isolated plasmid)
Hope maybe that helps a bit!

Hi, thanks for the advise, i did not make a typing mistake with the anneling temperature, the stratagene's protocol say that you can go from 60 to 68 C in this step, they do not mention lower temperatures, i am using 68 because at 60 i had only parental plasmid at the end. The concentration of the primers is 125 ng each... that is want the stratagene's protocol suggest.........
I am going to try it again, with new primers, and less concetration of primers and template, you think that could help???, do you think that i may need to increase the cycling times????

Try different template concentrations for your next run.

good luck !!!

Try to do the PCR for 40 cycles to see if you get any amplification at all. I would strongly recommend using 55°C as starting annealing temp and to increase your annealing time from 10 seconds to 30 or even one minute.
What e. coli strain is your plasmid isolated from (100% sure it's methylated?).
If you have accuprime pfx, you might try it, maybe you're more succesfull with this enzyme (though pfu's work very fine). Also: dilute your template, I never go above 30 ng for mutagenesis reactions, mostly I use 5-10 ng. Increasing cycles from 18 to 25 might also help.

Good luck.