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How to test my milleq water in q PCR about contaminants

qPCR probe primer water

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#1 Luiz Fernando Lino

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Posted 12 August 2014 - 05:56 AM

I will use my mille q water  for probe and primer diluition, I will work with DNA.

 

so I'm very worry about the contaminants, how can i have this water quality control. Run a convencional PCR and a policrilamide gel?

 

thanks



#2 Trof

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Posted 23 August 2014 - 09:30 AM

You can autoclave Milli-Q water in autoclavable glass flasks if you want to, but many bioforum users afaik use Milli-Q directly. If your system is cleaned regulary and you don't contaminate it while out of the fiter, you can hardly get PCR contaminants there.

 

But for checking just do a negative control (PCR setup for some well working DNA assay mix with primers with water instead of template), and do it with every other assay you run. I don't know what is your custom, but usually we run PCR on agarose gel, not acrylamide, but that probably doesn't matter as much.

 

But that can't tell you if you have a nuclease contamination in your water. You would know that, if your DNA will be degraded, instead of single band on 0.7% agarose gel, you  will see a smear and your PCR would be failing. It can't also tell you about PCR inhibitors contamination, that would also make your test PCR fail.

But again, that is unlikely to hapen with Milli-Q water.

 

So make some PCR with positive template and without template and if you got a product in positive sample, and no product in negative, you are probably OK.


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#3 hobglobin

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Posted 23 August 2014 - 11:11 AM

But I'd avoid autoclaving the water since this won't remove possible contaminants (i.e. DNA) but might add new dirt.


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#4 Trof

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Posted 25 August 2014 - 02:04 AM

hobglobin: I would say incorrect handling during autoclaving can add new dirt (or dirty/rusty jars used for plastics for example). Or if the autoclave is really nasty. Anyway it's better to put milliq water into clean glass flask, that has been autoclaved before use. While the water is extra pure in the MIllipore machine, the containers you put it in, often are not. It's a matter of decision to autoclave it after, since of course autoclave won't get rid of DNA or even ions, but may get rid of potential "living" contaminats that you introduce to the water while handling it in the open air.


Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon






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