I want to prepare solutions with nuclease free water to run RNA samples(isolated from Mirvana isolation kit) on denaturing acrylamide gel. It is given in protocol not to use DEPC treated TBE. So , what is the best way to make nuclease free water in that case?
Add 0.1% DEPC to MilliQ or Double Distilled water - let it sit overnight at 37degC and then Autoclave it. Make sure the glassware used is also rinsed with the same water or treated with Chloroform or Baked in a hot air oven (260degC) for 4hrs. It should be ready for use - both DNase and RNase free. For a detail understanding here is a link. Then you can use this water to make your stock solutions and eventual TBE. Make sure you use DEPC treated water to dilute your stocks as well.
http://protocols.mmml.nl/protocols/other/DEPC.php
Add 0.1% DEPC to MilliQ or Double Distilled water - let it sit overnight at 37degC and then Autoclave it. Make sure the glassware used is also rinsed with the same water or treated with Chloroform or Baked in a hot air oven (260degC) for 4hrs. It should be ready for use - both DNase and RNase free. For a detail understanding here is a link. Then you can use this water to make your stock solutions and eventual TBE. Make sure you use DEPC treated water to dilute your stocks as well.
http://protocols.mmml.nl/protocols/other/DEPC.php
You can also use DMPC. For me it wa smore convenient, because you can be sure that autoclaving once is sufficient
Regards
Sven
If you have Milli-Q water:
See this link from NEB about "Avoiding Ribonuclease Contamination": https://www.neb.com/tools-and-resources/usage-guidelines/avoiding-ribonuclease-contamination
"As an alternative to the historic use of DEPC, which can inhibit enzymatic reactions if not completely removed, we have found that Milli-Q™ (Millipore) purified water is sufficiently free of RNases for most RNA work."
@ Kalpana Sahoo,
Autoclaved Milli-Q water should be good enough for PCR use. We have been using that.
- Badges
- Science method