4 replies to this topic

[harri816]

Hi,

So my lab does a lot of PCR to create knockout constructs and gene tags, and over the past couple years we have had intermittent problems with contamination.  Many of these problems we have narrowed down to be due to the water we use in the reactions, and consequently have tried a variety of different options, including DEPC-treated water and Type 1 Ultra-pure water from a Milipore Mili-Q filter.  I was wondering if anyone else has had similar troubles with a given type/source of nuclease-free water in the past and what you have used with success.

Thanks!

[Trof]

We never had such problems, but usually use PCR water from the mastermixes we buy. You can buy even commercial nuclease-free water, it's not cheap but you don't need liters for PCR and it's better that to throuw out whole reaction if you know you have problems with filtered water.
Our MiliQ is always autoclaved, but many manuals point out to autoclaves as a source of contamination.

[gebirgsziege]

Depending on your application, you should buy water for DNA extraction. I never had issues with milliQ water - besides cross contamination which happened during handling of the water.
So I always use small aliquots which I make immediatly after filtering the water and keep them in the freezer.

[LpBio]

You can try to prepare DEPC- water in the lab. I ve been using it for PCR and got no contamination in my negative controls ever since.

[Trof]

Problem with DEPC water is, that it can has pH as low as 5. There is always buffer present in PCR reactions, but I'm not sure how big is the buffering capacity when large volumes of such water are used.