chronic PCR contamination - (Mar/10/2006 )

I am having severe PCR contamination issues that have been going on for about 6 months.
We have gotten all new primers and reagents several times now and it is getting very strange and frustrating. We have cleaned all the pipettes and benches. I am the only person in the lab who has this issue and it is not a technique problem (i.e. hitting the pipette tip with dna on it accidentally into the negative reactions etc). I have to set up all of my reactions in a separate room in a hood. I have even set up a pcr that is totally a negative control, did not even take any dna out of the freezer and I had bands all over the place.

Also, we are working on two different genes and this issue only comes up when I work with the primers from one gene versus the other. Working in the hood helped for a few weeks and then yesterday I got a very strong positive in my negative even when in the hood. I even wear two pairs of gloves. I dont know what else to do, I am lucky to be working with wonderful patient people, but I am going crazy and feel like some kind of a freak. Someone else will set up a reaction and it will be fine and then I will set it up and it is contaminated. Any suggestions would be greatly appreciated.

How good is your water? Are you autoclaving your tubes, or using from-the-box? Autoclaving the tips? What species is this? Taq often has coli contamination from genomic DNA. Can you use UNG and dUTP containing dNTP mixes?

I would recommend that you buy pure water intended for PCR, and stop autoclaving everything (tips, tubes, water). Instead, buy RNAse/DNAse free tubes and tips. You can buy them sterile if you need them to be, but this is usually unnecessary. Most autoclaves I have seen add more contaminants than I would like to see in my PCR reactions.

A student in our lab had a similar problem, and it dissapeared after we started buying clean tubes/tips and water rather than autoclaving them.. so i would do as phage434 suggested.

Also, this may seem redundant but are u amking sure not talk when you are in the hood, and make sure u are not passing yr hands/arms over open tubes and tips?

I can imagine its all very frustrating... good luck with the trouble shooting

in my lab another student had the same problem:

he solved cleaning the hood and pipettes at first, then using gloves (one pair is enough), celan coat and a lab mask to avoid contamination.

And, of course, tips, tubes, water and reagents have to be for the hood ONLY (do not leave them on the bench and then use it for the PCR).

check TAQ, for contamination, too...

I know it can be very hard and frustrating. In the lab I was before we had similar situations. It is not very scientific, I know, but sometimes the contamination problems dissapeared stoping to work in the issue for 3-4 weeks and coming back to it after.
Also heard about possible contaminations coming even from centrifuge, from really experienced people. I would use water and reagents from people in your lab who are doing PCR and not having contaminations. Good luck

I switched to sterilized water that we purchased instead of autoclaved, cleaned the hood and everything in it and even my lab coat and I will see how it goes. Thanks for your help.