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DEPC-water and DEPC-PBS


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#1 Samantha

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Posted 26 March 2005 - 11:05 PM

For RNA extraction, we have to make DEPC-water and DEPC-PBS. I want to ask how do you guys usually prepare these solutions?


1. clean and autoclave the empty bottles
2. fill with 1 L NANO water and add PBS tablets for DEPC-PBS
3. add 1 ml DEPC to DEPC-water and DEPC-PBS respectively
4. shake the bottles at 250rpm at 37C O/N
5. autoclave

Do you guys do the same, especially autoclave the bottles first before filling with NANO-water? I have this question because some of my labmates argue why we have to autoclave the bottles for two times, but the other labmates say RNA extracted was degraded when they didnt pre-autoclave the bottles. Do you have the same problem??


samantha

#2 littlecell

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Posted 28 March 2005 - 07:18 AM

i used to autoclave the glass bottle by heating it at 260 centigrade for at least 6 hours, then following your steps. but i think you had better purchase DEPC-treated Water for RNA ISOLATION from QUALITY BIOLOGICAL. It works very well. so why trouble to prepare it by yourself?

#3 fred_33

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Posted 28 March 2005 - 08:09 AM

hi
in my lab we let DEPC trteatment for at least 24 hours in a non pre autoclaved bottle. And non particualry degradation if extraction done quick and on ice.

#4 lula

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Posted 29 March 2005 - 08:07 AM

hello
for the DEPEC treated water of RNA extraction, we put the depec on the nano water then leave them for 24 hours (the water we use is autoclaved before adding the DEPEC)
after adding the DEPEC we leave it for 24 hours then autoclave again

#5 ajames

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Posted 29 March 2005 - 10:01 PM

I wouldn't bother DEPC treating water. Too many things can go wrong-residual DEPC, incomplete inactivation and the fact that it is a neurotoxin. There are lots of suplliers of nuclease free water and this will not be a significant part of the cost of performing PCR. Why not buy the water in the same way you buy the taq? The PCR will only be as good as your worst reagent.




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