Hello everyone
Can I use double distilled water(ddH2O) to make a high volume PCR Master Mix ??? DO I have to use DEPC treated water (ddH2O) to make PCR Master Mix???
Unfortunately, I do not have access to DNase RNase free Water and MilliQ Water.
Thank you in advance for your help.
No problem. You can use autoclaved ddH2O if you are just concerned about DNase; however, if you plan to do RT-PCR, for which integrity of RNA is important, you will be safer using DEPC-treated water. Treat with DEPC first, then autoclave, which will get rid of DEPC.
Pierre Béguin Thanks for your answer
According to the following protocol:
· DEPC is used at 0.1%
· Thus you would typically add 1ml of DEPC to 1 litre of double distilled water
· Because DEPC is hazardous you perform this action in a fume hood, stir in the DEPC to inactivate RNASes over night
· In addtion incubate the next day for 12 hour 37C to facilitate RNAse killing by DEPC (optional)
.Autoclave DEPC-mixed water for 15 minutes or 45 minutes.
If you use use DEPC it is hygroscopic - that is abosrbs water dilutin it - so should be stored in the dark with parafilm around the lid and replaced every 2-3 months
To autoclave DEPC treated water, do I have to close the glass jar tightly? And after preparing, should I keep it in the dark and at room temperature?
hi,
I also used filtered deionized water and there was no significant difference, as long as the total process time was less than 12 hours.
Note that using DEPC is the best and only option
I, like most people in my lab, use autoclaved ddH2O for all of my regular PCRs and so far had no trouble whatsoever.
Hi
The use of distilled water is used routinely for PCR but can be better decided based on the type of test and the type of target, if you are working on RNA it is better to use DEPC.
In addition, “PCR water” is ion-free (i.e. distilled H2O) as a way to minimize interference with the activity of the DNA polymerase. Zahra Dorosti
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