false positives in PCR due to environmental contamination? - (Jul/16/2008 )

I'm struggling for past 2.5 months with RT-PCR contamination- getting false positives. So I got fresh primers yesterday, and I decided to run a no-template (only primers with PCR master mix). We have a laminar flow with UV which we use to passage animal cell lines (only cells; no infection or handling bacteria). But the point to note is that we have three other laminars in the same room where cell infections are carried out. I wiped the surface with 10% bleach, 70% ethanol, and let UV on for 15 minutes. I UV'ed my tips, gloves, tubes, rack and even the ice bucket for 5 mins in heavy UV. I then wiped the surface of each of these with bleach and ethanol, and placed them in the hood. I brought my new PCR master mix, aliquoted in PCR tubes, and finally opened the new primer one at a time, and added to the master mix. I wore a sterile lab coat (the disposible one that's used in surgeries) and changed gloves when I was going to handle the primers. I STILL got a faint band, of the expected size of the genes.
I then requested one of my colleagues in an another lab to do a single step RT-PCR (I use a 2 step RT-PCR for my experiments), and gave him the RNA and the same primers I used earlier. His negative controls were perfectly negative!! Do you think this is environmental contamination or a problem with my handling?

Thanks for the response...I did try that.. the pipette I used yesterday was a set of pipettes used only for preparing primers... Its never seen a DNA larger than 100bp. And the PCR master mix I used yesterday was new... never opened. and the water was fresh (i mean fresh from the milliQ machine).

Perhaps it is environmental (been known to happen). Ask your friend if you can set up your PCR in his lab. Might seem troublesome, but hey, if the problem goes away then it's worth it.

Use also pcr-grade water and (presterilized) filter-tips for the pipettes (do NOT autoclave them again!!!).
It also depends on your target dna/rna you want to amplify with your pcr.
if it is human the millipore water can be contaminated - always use commercial pcr-grade water - it is really inexpensive compared to masters, primers, etc. and very good tested...
If you amplify bacterial dna/rna remember that all of the pcr enzymes (usually recombinant) used for molecular biology are purified from bacterial cells (usually E. coli) and together with the enzyme most of the time small amounts of nucleic acids are co-purified which later can contaminate your pcr rxn.
Use a master / enzyme from a well known pcr-supplier e.g. Roche or ABI, they have special procedures to make their enzymes nucleic acid free...
Never use the same room or pipette for pre- and post-PCR procedures (i.e. preparation and running the gel etc.)...
...sometimes it helps to go to another lab and use their pipettes AND plastics...
...sometimes contaminations come from the autoclave... because dna is not destroyed inside.. but can be spread with the steam from an old pcr rxns all over the plastics, pipette tips, solutions, water,.... which is also in the machine...
... hope one of the possibilities fits your problem...
...finally I wish you all the best

one addition:
...do not use etoh - it doesn't kill your dna - why should it?
maybe you can use "dna-zap" or "licence to kill" or similar for decontamination in the future...

thanks for the reply senior scientist... you know whats frustrating? nobody in the lab believes that pre- and post PCR operations are going to do me any good.. they see me like speaking based on imaginary things... even my boss doesn't seem to understand... as smu2 said, am going to try doing my PCR in the other lab and see what happens...