Depc

[Jose]

Until now, i usually use DEPC- water, and treated matherial (tips, tubes, glas...) to work with RNA. But I see other labs dont work so.
It is possible not to work with DEPC? What do you think about it?

[moleculardude]

Dear Jose,
welcome to the forums!

We are one of those labs that do not use DEPC. It is very possible not to use DEPC water, however you must take certain precautions.

If you use RNase inhibitors such as Promega RNAsin, then you really do not need to take many precautions.
Even then, I use Milipore (rnase free) water for making buffers and solutions.

If you do not use RNase inhibitors then you must be very careful to use clean, sterile glassware AND milipore water (rnase free).
If you use regular distilled water, you may have some problems.

Cheers!
Moleculardude

[nkaur]

is it necessary to sterilize pippets.If it is then how it can be sterilize?

[pooja]

hi all,
if u are not using depc i ur lab. then
plz. let me know what are those precautionary steps that u r foliowing.
as am extracting dsRNA in hood but am not getting any result.
i thought u it may be depc, if u recommend MQ water by that u mean
what autoclaved or not as i heard autoclving spreads rnases.
my mail i.d is poojaapr1@gmail.com

[deltafx1942]

moleculardude, i was under the impression that treating glassware/plasticware with an RNase inhibitor like RNase-Away was the main precaution to take when working with RNA, besides the usual like gloves.

what other precautions should one take when working with RNA?

[̑̑̑]

always use either depc treated reagents, or use rnase inhibitors, treat plastic/glassware by sumerging o/n on 0.1% SDS and rinse next mornig with rnase free water and then etahnol to remove traces of detergents. use sterile tips and never autoclaved them as water vapour in autoclaves is full of rnases. wear gloves and use your common sense it will take you as long way in the lab... unless your common sense is bush-like lol

[pooja]

thanks to all, i get through my problem but the band of dsRNA which is coming out is comparitively faint means the yield is low. can i improve my results further. what do you suggest?
thanks once again

[pooja]

and now, its been very true that using depc is not essential, M.Q water and some major precautions for RNA can work out the problem.

[moleculardude]

I definitely use autoclaved pipette tips all the time. Yes this is a must. I am of the school of thought that DEPC and baked glassware do not need to be used ONLY when using RNase inhibitors (and plenty of them!)

Saves time (but not money)...