From 5-5-15.
I performed diagnostic PCR on the five P. larvae isolates using the AFB primers using new PCR super mix. However, the ran gel was completely blank except for the ladder! Even my positive control, P. larvae 9545, did not have a band appear. I continue to have issues with my PCR reactions.
I don't believe there is an issue with my DNA template, since my positive control has been used successfully in the past. However, perhaps the DNA has began to degrade and is no longer viable as a template for the reaction. But that doesn't explain why none of the P. larvae isolates amplified.
There may be an issue with the PCR supermix. We just received it in the mail and I have never successfully used it before. Perhaps I am not mixing the reaction correctly, however I did heavily scrutinize the instructions and am fairly confident the reaction was set up appropriately. I can ask my lab mates who have also been using the new supermix if they've been having any issues with it.
Maybe there is an issue with the thermocycler itself? As mentioned, this is not the first time I've had issues with PCR recently. I double checked my parameters for the AFB reaction and it is still set up appropriately. But, perhaps the thermocycler is having an issue with getting to the temperatures that are listed or maintaining that temperature.
I will further investigate these possible phenomenons as a possible source of my PCR problem.
//EWW
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