All PCRs in lab stopped working - We all use different reagents - ideas? (Dec/03/2008 )

Hey guys, sorry I've been posting so much, but for the past eight days everything has failed. Any idea why ALL of our PCRs would stop working? Including colony PCRs, reverse transcription PCRs, PCRs with different primers and recipes, one fish two fish red fish blue fish?

At first only our undergraduate had the problem, but now none of mine will work. Today I got a kit with a control PCR setup and that didn't amplify either - everything came with the kit, the only thing I added was the Taq and water.

If the water's contaminated, I would expect to see some amplification, not nothing at all. And the Taq came from a different lab that has no problems.

If it's just the reagents (buffer, dNTPs, primer, whatever) then the control PCR setup should have worked.

If it was the thermal cyclers, which are shared property, then the whole building would have problems... and I've tried three different machines.

The cycling conditions worked before. Now they don't.

There are just absolutely no PCRs happening here.

Also, all of the RNA preps I try to make have failed - there's nothing in them and they're degraded - which should be impossible because I'm using Trizol on some of them and Ambion RNAqueous extraction kit on some of the others and then I run it on a gel and there is NOTHING THERE, like a tiny low molecular-weight smear.

Could this be a function of the lab (like contaminated benches) or the equipment (all of our pipettes are haunted) ?

Or maybe it is just creepy

Cheers!

Hey guys, sorry I've been posting so much, but for the past eight days everything has failed. Any idea why ALL of our PCRs would stop working? Including colony PCRs, reverse transcription PCRs, PCRs with different primers and recipes, one fish two fish red fish blue fish?

At first only our undergraduate had the problem, but now none of mine will work. Today I got a kit with a control PCR setup and that didn't amplify either - everything came with the kit, the only thing I added was the Taq and water.

If the water's contaminated, I would expect to see some amplification, not nothing at all. And the Taq came from a different lab that has no problems.

If it's just the reagents (buffer, dNTPs, primer, whatever) then the control PCR setup should have worked.

If it was the thermal cyclers, which are shared property, then the whole building would have problems... and I've tried three different machines.

The cycling conditions worked before. Now they don't.

There are just absolutely no PCRs happening here.

Also, all of the RNA preps I try to make have failed - there's nothing in them and they're degraded - which should be impossible because I'm using Trizol on some of them and Ambion RNAqueous extraction kit on some of the others and then I run it on a gel and there is NOTHING THERE, like a tiny low molecular-weight smear.

Could this be a function of the lab (like contaminated benches) or the equipment (all of our pipettes are haunted) ?

Or maybe it is just creepy

Cheers!

maybe there's something wrong with the gels? did you check an old sample on gel together with the new samples? DNAses in your gel buffer/loading buffer might be destroying your samples ...

In our lab, none of the PCRs seemed to work, and we found that the problem was the newly prepared 10xTAE agarose gel buffer. The glycine conc. was 1/100 of what it was supposed to be, and the bands looked all messed up or nowhere to be found.

This problem sounds spooky! The MQ water could also be a problem? We had a period when the machine didn´t work properly, the pH of the water changed dramatically and was too low to elute DNA. Could interfere with PCR as well?

Thanks for all of your answers! I know it isn't the water, though; I've used a number of different kinds including our stock MQ as well as previously unused Nuclease-Free samples provided with various kits.

Moving to another lab area and using different pipettes didn't help. Neither did using a kit of reagents that a neighboring lab used with success.

I have tried controlled groups of reactions in which each component was replaced by a new one. I have also tried setting up control pcrs with unused materials that come with kits

The degrading gel idea was a really good one, but unfortunately I don't think that's the case - the ladders are fine. Chimp, what were the ladders like on your bad gels?

Really, any more ideas would be appreciated, I have no idea what to do. I can't get RNA to work and PCR just stopped working and these are simple things.

EDIT: Thank you all so much, by the way.

Thanks for all of your answers! I know it isn't the water, though; I've used a number of different kinds including our stock MQ as well as previously unused Nuclease-Free samples provided with various kits.

Moving to another lab area and using different pipettes didn't help. Neither did using a kit of reagents that a neighboring lab used with success.

I have tried controlled groups of reactions in which each component was replaced by a new one. I have also tried setting up control pcrs with unused materials that come with kits

The degrading gel idea was a really good one, but unfortunately I don't think that's the case - the ladders are fine. Chimp, what were the ladders like on your bad gels?

Really, any more ideas would be appreciated, I have no idea what to do. I can't get RNA to work and PCR just stopped working and these are simple things.

EDIT: Thank you all so much, by the way.

Do you add mineral oil to your PCR reactions? DNase contamination in the oil perhaps?