I'm genotyping a line of mice and am trying to create a PCR thermocycler protocol that uses 4 primers, 2 for the WT gene and 2 for the mutant transgene. I've run this a couple of times but can't get it to turn out. The primers have slightly different Tms:
- WT Forward: 66 oC / WT Reverse: 62 oC (297 bp)
- Mutant Forward: 65 oC / Mutant Reverse: 63 oC (253 bp)
For all the samples, only one band's really appeared and the other is barely visible and they're smeared so can't tell which one (WT or mutant gene) it is.
I've been trying a touchdown protocol with an anneal temp starting at 68oC (-0.8o per cycle), 11 cycles for the touchdown and rest of it anneal at 60oC, 24 cycles. I'm not sure if I should keep trying a touchdown protocol or if I should do a standard PCR with only 1 anneal temp and 30-40 cycles (I just don't know what temp that would be).
Any guidance and suggestions would be greatly appreciated! I can provide any additional info if needed. Thank you!
My advice is to set up separate reactions & not try to multi-plex.
I think you should get the results below first:
clear band with WT template and WT primers
clear band with mutant template and mutant primers
no band with WT template and mutant primers
no band with mutant template and WT primers.
Then, find a good TM works for all above.
then, try mix the primers and use either WT or mutant template.
I assume that your normal and mutant primer sequences differ by one base at ,or near, the 3' end of the primers. In this case if the touchdown to too low an annealing temperature it may allow the normal primer to anneal to the mutant sequence and the mutant primer to anneal to the normal sequence..
You could try setting up gradient pcr for all of the primer sets separately against both normal and mutated dna samples which will tell you what temperature ranges the primer sets can be used in without amplifying the wrong allele
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