Can DEPC-water interfere QPCR reaction?
Posted 17 February 2009 - 03:12 AM
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 -
Posted 18 February 2009 - 03:22 AM
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
Posted 18 February 2009 - 12:09 PM
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?
Posted 19 February 2009 - 10:29 AM
Posted 27 February 2009 - 08:25 AM
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.