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Can DEPC-water interfere QPCR reaction?


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4 replies to this topic

#1 Thana

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Posted 17 February 2009 - 03:12 AM

I have strange QPCR results lately and I'm assumping that there's contamination in my DEPC-water which is also used to prepare the Mastermix.

However, my supervisor doesn't think that it was from DEPC-water; It's definitely RNAse-free water as the same water was used in all

preparation (RNA isolation and DNAase step) and RNA ratio was high.

And I've checked primer and probe and there seemed to be nothing wrong with that !!

What else could go wrong in the reaction as everything is kits but not the DEPC-water?

Can someone help me? All the suggestion would be appreciated.

- Thanks -

#2 molgen

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Posted 18 February 2009 - 03:22 AM

In one word?
Contamination!!!

You might have a contamination in your samples/tubs/pipets…
Try working in a laminar hod, use filter tips and so on.
DEPC treated water can get contaminated after the autoclave by bacteria and algae. Always dispense it to aliquots in the hood

#3 wuxx0153

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Posted 18 February 2009 - 12:09 PM

Another possibility I know of is PCR inhibitor in your RNA samples.

If your PCR inhobitor is lipid or carbohydrate (last time our inhibitor is carbohydrate), they will not change your OD reading a lot, but for sure will affect your PCR reaction.

What is the source of your RNA ceom form?

#4 tea-test

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Posted 19 February 2009 - 10:29 AM

Maybe there was some DEPC left in the water? I always use for RNA and PCR molecular biology grade water from Invitrogen cat.no. 10977035. it's only 12 EUR for 500ml. never had a problem and i also don't have to hassle around with the toxic DEPC...
tea-test: The artist formerly known as Ned Land

#5 ra_polymerase

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Posted 27 February 2009 - 08:25 AM

It does sound like contamination--remember that DEPC only inactivates RNases that are present in water when its made, and won't inactivate RNases that are introduced after its been autoclaved.

Residual DEPC that hasn't been inactivated after autoclaving can also inhibit RT and PCR reactions (or so I've heard).

Use Ambion's Nuclease-Free water, I always use the small 100ml bottles of that for qPCR and have never had problems.




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