I am trying to confirm the presence of bacterial DNA with some pretty robust primers. I am using a very simple DNA extraction protocol (just boiling the colonies!) but am having trouble getting consistent results. If anyone is willing to look at some gels, please PM me and I will happily email along the photo.
I am using a primer for the 16S region (nothing specific for staphylococci - just a generic bacterial primer) to reduce my false negatives. I have taken colonies of staphylococci (grown on selective media) and placed them in TE buffer, boiled them at 95°C for 10 minutes, briefly centrifuged for a few seconds, and then taken 2.5 uL of supernatant and added it to my PCR reaction.
Last week I performed PCR on DNA from the same 96-well plate. There were several differences in the two reactions so troubleshooting is a little difficult. The first PCR I performed resulted in 69/96 bands - lower than I was expecting, but manageable. Interestingly, it was clear that columns 10, 11, and 12 had absolutely no amplification so it seems like something is up spatially. I then performed PCR a second time in an attempt to remedy this. For this, I went back to the 96-well plate that had the colonies that had been boiled once and briefly centrifuged once. I again boiled the colonies and I again briefly centrifuged them. I boiled the colonies in a different orientation on the thermocycler since I have my suspicions about that particular piece of equipment. I then also used a different stratagene for the PCR. This time, I only got 33/96 positive -- but interestingly, almost all of the samples in columns 10, 11, 12 resulted in a band.
Clearly, these results are too inconsistent to be useful for anything and it is also really not clear what could have caused the inconsistencies. I mix all of my reagents in one reservoir and then multichannel pipette them into the centrifuge plate so it's not clear why one section of my plate would be any different than the others in terms of human error. My suspicions was that some of the wells of the boiling stage did not get hot enough, but that does not explain the fact that nearly 75% of the heating block worked fine the first time but only ~25% worked the second time.
How would you approach troubleshooting this?
Thanks so much.