I have a plasmid with 9kb length, I am using DMSO since I heard it increases the efficiency. The PCR protocol I often use is as follows with various annealing temps such as 55, 60, and 65C;
95C 1 min
95C 50 sec
55C 50 sec
72C 5 min
16 cycles
72C 7min
I am pretty sure my PCR does not work. I get colonies, but sequencing results show wild type plasmid. Could you give me some tips about any part of SDM protocol or the material that I used?
First, 72C 5 min could be the issue for a 9kb plasmid. Usually its better to calculate 1min/kb size as extension time, irrespective of standard enzymes used.
Second, Dpn1 digestion is key to get rid of wild type ones. 1hr should be good enough.
Transform using NZY+ broth
Finally, 9kb plasmid needs some luck at every step.
This brochure has every details you need
https://goo.gl/QFH9li
Are you using two primers for mutagenesis? Are they exactly the same? Try using 65 or 68C for 8-9 minutes. 1-2 hrs DpnI digestion is a must. I would rather do 2 hrs than 1 hr.
Or are you using single primer mutagenesis protocol?
Thank you for the tips. Yes, I am using complementary primers. There is this new protocol I learned from a colleague if anyone is dealing with the same problem. You do the annealing temperature as gradient and perform 25 cycles, then do dpnI digestion for 15 hours. When I ran them on agarose gel, there were clear bands and dpnI worked just fine. I am waiting for sequence results.
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