I have known researchers to use nanopure water from an in-house system but it was only for eukaryotic work. Generally speaking, as mentioned above, most nanopure systems are not cleaned thoroughly enough to avoid bacterial contamination (which is fine for most of us). Even with my ultrapure nuclease-free water, I separate it into 1mL aliquots, irradiate the tubes, and then use it only once or twice before re-irradiating or discarding the remainder. If you really need to use your nanopure system, you might try filtering it using a new 0.2 um syringe filter after getting it from the machine, and then irradiating the filtered water. Still, if your water source is suspect, best practice is to get a reliable source. It may save you money from not wasting time and experiments in the long run.

We use the Sigma's pcr grade and molecular biology grade H2O for 16S PCR and qPCR. It seems to work quite well. The best info we have on those is what is available on the website: less than 10 cfu/mL for the mol bio grade (which is prob the limit of detection of the assay).

ddH2O is no good, and most routine use Mili-Q isn't much better (the better quality systems, which produce water for analytical chemistry and such, work well). Pseudomonas (as mentioned) is incredibly prevalent in dust everywhere. Good practices are definitely a must. We work on TC hood and have all your consumables UV irradiated.

Best.

We usually buy water from invitrogen/gibco for our prokaryotic PCRs, however it is expensive all things considered.

Both I and my colleagues have had success using millipore filtered water which has been autoclaved, and we usually make DEPC water in-house even though it is a little time consuming.

I agree with Seweryn - not even the most sparkling clean water is going to save your PCRs if you do not have appropriate contamination controls in place! and water is the cheapest reagent - so replace it frequently!

Thanks! I just started working in a hood, using the germicidal light, and my PCRs have been working better. It hasn't mattered in the past, because we were not doing detection limits, and we were only doing 20-30 cycles. Now I am working on detection limits with 40+ cycles.

We have been following the norm of irradiating hood with dedicated plastic water and pipetter with UV for atleast 10 min before the work. we clean the working area with 70% alcohol. The PCR area is separate and nothing is brought out of the hood. With this convention even though we use millipore filtered MilliQ, and autoclaved tips and tubes, we have not found cross contamination till now. So working conditions surely help in a good PCR lab.

Good technique is definitely a must (and usually saves a lot of headache on troubleshooting anyway).

But PCR negative controls only ensure the PCR was set up properly. Environmental contamination during DNA extraction of your samples is also a concern, most specially when the samples have minute amounts of material to begging with.

In our lab we always carry out blank extraction controls side by side with your samples. They become a "extraction control sample". This is specially important if you are pushing the limits of detection (e.g. 40+ cycles or qPCR), so you can rely that the small amount of material you are measuring is really important, and not just dust wondering into your tube.

We have used several brands of "molecular biology grade" water for our broad-range bacterial PCRs, and got good results with all of them, as long as they are nuclease and nucleic acid free. We are currently using the Thermo Hypure brand, which works well. But, as you already noticed, other measures are equally important to keep your negative controls, well, negative. These would include physical separation (different rooms, different sets of pipettors) of pre- and post-PCR steps, using filter tips, and UV-radiation of pre-PCR hoods. Even then, we sometimes run into contamination problems. Polymerases are made using bacteria, and they sometimes contain bacterial DNAs, PCR additives (BSA, DMSO) can be contaminated, and certain broad-range primer combinations appear to be very sensitive in picking up some background DNA, despite following all the measures suggested above. I hope this helps!

MilliQ water from millipore, sterilized in autoclave doesn't contain DNA rests. If you fill yourself the boxes of tips for the pipette, do it with gloves and also sterilize in autoclave. Good luck

hi! I am doing DNA extraction from the skin swabs to identify Staph aureus, and I used 3 negative controls: just water+ extraction kit, just extraction Kit and empty swab. My DNA showed low values ( around 7ng/ul) but with 260/280 with negative values (-11.2 for example). My samples showed a small amount only since skin swabs are a little hard to get some DNA (around 13.5 with 260/280: 4.03). the same batch I used oral swabs (23.4 ng/ul with 260/280 2.1). So I am a little confused, my water and the kti are contaminated? What this negative value means? thank you for any help!