I have been working to generate a protein domain deletion for some time using
QuikChange II Site-Directed Mutagenesis Kit by Agilent. I am not able to get the amplification either in test or control. Though, primer dimer band is visible. The protocol is being followed properly. The template size is approximately 8.4 Kilo bases, the G:C content and the length of the primer is also high. I have tried to play with different conditions and reagent concentrations as follows-
(1) primer concentration: 100uM,150uM, 300uM, (2) template concentration: 100n, 200g, 250 ng, (3) polymerases such as phusion, agilent pfu and Q5, (4) Annealing temperature- 55,57,58 degrees, (5) Extension time- 10 mins and 20 mins, (5) Addition of DMSO or formamide in the reaction mix.
Any suggestions to troubleshoot would be highly appreciated. Thankyou.
Yuhta Nomura The concentration mentioned above is for 50ul of reaction mix. I forgot to mention in the question. Relative to the normal PCR mixture, it is recommended to use higher primer concentration in SDM to obtain desired mutant amplification.
I will like to ask about the amplification. No amplification on either the test or the control. Surely you would like to see a control where no amplification is used? I will like to suggest the kit itself. Or the approach to the amplification, or the specimen. Agilent is good, does it say not to use an enzyme? the pfy agilent is also effcient.
If you say it is recommended, I will not say, but I would mention the first comment.
Perhaps I will add the template. Is not 8.4kb too high? Surely you must see a trend after soo many conditions?... Maybe then say it is the kit or the approach.
I will add the reagents like DMSO, but maybe it is not relevant. It is not as Mg+2 and polymerases. I don't think the polymerases are relevant.
Hope it helps.
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