We have been trying to run genotyping protocols. We ran a gradient PCR to re-optimize the annealing temperature, tried to change the ramp speed and also modified the DNA purification protocol. Has anyone faced this issue? Would appreciate any advise on how to get this working!
go conservative on all parts of the cycle until things are working....lower annealing, higher denaturation and longer times. Your gradient tells you that the annealing temperature is not the problem so more likely it is the denaturation temperature has changed. Run pcrs with other primer sets and with reagents that do work on another machine just in case the regents/primers have degraded and use dna that has amplified before. Are you sure that the machine is cycling?
Try a cycle of 60c then 4c and run it touching the block and see if it feels hot/cold at the appropriate times. Check the expected total cycling times eg if the pcr 30 cycles should take 2 hours then you have a block problem if it takes 4 hours.
Read the instruction manual especially the programming and troubleshooting sections
Investigate particularly anything that has changed at changeover time. Another possibility is that the plastic tubes/plates/strips that you use may be thicker than the recommended ones and be insulating the samples or that the volume is larger so more reagent mix evaporates.Try a thinner tube
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