inconsistent results - easy 16S amplifcation is not so easy - (Feb/08/2010 )

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.

Attached is a picture of two gels with the same clinical samples. These both came from the same 96-well plate. There were several differences in the two reactions so troubleshooting is a little difficult. The picture on the left was on the PCR performed first so the protocols were exactly as I mentioned above. The picture on the right is from when I attempted to repeat the PCR since I was confused as to why none of the lanes at the bottom of the gel turned out. 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.

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?

What is the PCR product being used for that makes these "result too inconsistent to be useful"?

Many of your wells seem to be overloaded with template DNA. When I do colony prep PCR with 16S rRNA I only use about 1ul of cell suspension (and even this can be too much sometimes). I also work with Gram positives (filamentous, spore formers) and don't have to heat the cells to get a product from PCR. Just add water and colony, vortex and add to PCR.

Also.... given the shadow that is over the bottom part of your gel, did you observe the gel directly or only through a camera lens. I have had problems with bands at the edges of large gels not being visable when images are captured but are present when observed directly.

Cheers
M