Nuclease free water for cloning - (Jun/04/2014 )

Hallo all,

Just curious to know if you all buy nuclease free water when doing cloning work?

I want to anneal some oligos in a plasmid and I checked some protocols and they often mention to use nuclease free water. I was just thinking to use autoclaved MQ.

It seems a bit overkill to buy nuclease free water for this type of cloning?

For working with RNA I get it, but for cloning?

No, I don't.  You might want to resuspend your oligos in nuclease free water, but it isn't absolutely necessary.

MilliQ should be fine.

We have MiliQ autoclaved water for general usage and DEPC-treated autoclaved water for RNA, but Qiagen (or any other) kits we buy come with an "RNase free" (supposing DNase free too) water, which should be ultrafiltered instead of DEPC treatment, so I actually prefer using it for RNA. And it's aliquited into small tubes, that's handy. So I use this for PCR, restrictions, everything.

So in the end we don't buy special water, but always have some spares.

Trof on Fri Jun 6 15:33:06 2014 said:

We have MiliQ autoclaved water for general usage and DEPC-treated autoclaved water for RNA, but Qiagen (or any other) kits we buy come with an "RNase free" (supposing DNase free too) water, which should be ultrafiltered instead of DEPC treatment, so I actually prefer using it for RNA. And it's aliquited into small tubes, that's handy. So I use this for PCR, restrictions, everything.

So in the end we don't buy special water, but always have some spares.

We have a lot of kits here too, but we dont have that much rnase free water to be honest.

You use the rnase free water for PCR and restrictions ? So in general you dont use autoclaved MQ?

I Always thought that dnases would be killed by autoclaving and that it were the rnase that are harder to heat inactivate.

So I figured that nuclease free water is not that really needed for PCR work or restriction reactions.

I would definitelly use MiliQ without problem for restrictions or PCR if I need to (not for RNA). We just now have the surplus, so if I can have a higher grade water, why not use it ;)

Thing is we autoclave our MiliQ water in 1L bottles. So right after autoclaving it's sterile, but if people are using it for several weeks.. you just can't be sure. For this reason I always took a just-autoclaved bottle, opened it in the flowbox and made several dozens of 1ml aliquotes in epp tubes, for my use.

(of course this water is used in all parts of the lab, often in huge quantities, inside and outside the hood, so when you need 50 ml you won't use 1ml aliquotes, but for PCR, RE, small-scale I find it the best solution, to use my aliquotes)

Trof on Fri Jun 6 15:33:06 2014 said:

We have MiliQ autoclaved water for general usage and DEPC-treated autoclaved water for RNA, but Qiagen (or any other) kits we buy come with an "RNase free" (supposing DNase free too) water, which should be ultrafiltered instead of DEPC treatment, so I actually prefer using it for RNA. And it's aliquited into small tubes, that's handy. So I use this for PCR, restrictions, everything.

So in the end we don't buy special water, but always have some spares.

How about milliQ autoclaved water's pH? have you had problems with it?

What's wrong with the pH?  MilliQ water usually has a pH of about 6 as it is deionised.

You should definitely not rely on the pH of milliQ water in any reaction. This is why you add buffers. High purity water has very variable pH, depending a great deal of whatever the last impurity it happened to come across. Low pH is common as a result of carbon dioxide in the air dissolving, but this can easily be overwhelmed by bits of detergent or whatever.  Use buffers!!