Touchdown PCR issues - (Jun/23/2010 )
I've develped a DGGE primer set for the genus Vibrio. It amplifys the desired product very well, but I asd getting unspecified binding that was higher in the gel. I then switched to a touchdown protocol, but now I'm not getting amplification of my product. Does anyone have a suggestion for me to try. The annealing temperature where I get the best amplification is 55C. Thanks!
Could you give details of you primer Tm's and the thermocycling parametres you are using for your touchdown PCR?
Well, the melting temp of one primer is 61C while the other is too high because it has a huge GC clamp on it. I performed a temperature gradient, and 55 and 56 anealing temp gives me the best amplification, but also gives me unspecific binding.
The touchdown pcr anealing temp starts at 65C and each requring cycle drops -0.5C for 20 cycles. Then 10 more cycles are performed at 55C. The hot step is 94 and the elongation step is 72.
I just re-read you original post and noted that you say the non-specific binding is higher in the gel. Usually this is caused by things like the extension time being too long, too much enzyme or primer. You may want to try looking at these factor.
But as for the touchdown thrermocycling I would try increasing your touchdown starting temperature (upto maximum of 72oC) or alternatively only decrease your touchdown temperature by 5oC (rather than 10oC) starting at your original 65oC.