I'm doing analysis on a DRD2 SNP for ADHD patients, with Touchdown PCR and electrophoresis. For the last 3 weeks I had to work on another PCR machine and now the Touchdown PCR doesn't work at all with the new one, even if the mix and the program are the same. Ideas? Analysis was done with the Qiagen kit for PCR analysis.
PCR instruments differ in several parameters such as amount of time to go from one temperature to another, and plates could be from different materials (platinum, ..... )
it could be that this new PCR is worse than the one you use before, I would increase anneling and extension times..... , it may help
Thanks for your help, I'll do new tests with your recommandations :)
Yes, I agree with Gloria. The machines make a lot of difference...you may get the results consistently on e one machine and never get that amplicon on other machines. This happens more often with dominant markers too.
Please, try again with one degree change in annealing temperature as some blocks heat up earlier and maintain while other blocks take time to heat up. So, it s only my little suggestion to change the annealing temperature
Thanks a lot for all your advices, I've tried and now it's working :-)
Thanks for all :-)
- Badges
- Science method