Touchdown PCR issues
Posted 23 June 2010 - 01:03 PM
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!
Posted 23 June 2010 - 02:28 PM
Posted 23 June 2010 - 07:40 PM
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.
Posted 23 June 2010 - 08:50 PM
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.