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Store restrict Enzyme in working system - (Jul/25/2008 )

Hi, ya'll. I have been doing PCR mutagenesis these days, and I have been doing this a lot. and I always have to double RE digest the plasmid for ligate or insert check with the same REs and same system. Can I just prepare the whole RE digestion system in the form of working concentration in batch then store them in -20C?

my system is like this:
BamHI 1ul, SalI, 1ul, NEBuffer-3 2ul, BSA 2ul, plasmid 6ul, H2O 8ul. (Enzyme, Buffer and BSA are from NEB Inc.)

Thanks in advance...

-lee_shore-

I don't think so, certainly not for long period. But you could try and report the results back happy.gif Without sufficient glycerol the enzyme will become inactivated when it is frozen.

What you can do is make up a master digest mix containing the NEB buffer, the water, and BSA. The restriction enzymes are added to the master digest mix just before use.

-perneseblue-

QUOTE (perneseblue @ Jul 27 2008, 12:08 AM)
I don't think so, certainly not for long period. But you could try and report the results back happy.gif Without sufficient glycerol the enzyme will become inactivated when it is frozen.

What you can do is make up a master digest mix containing the NEB buffer, the water, and BSA. The restriction enzymes are added to the master digest mix just before use.


Ya, what you said make sense. I just forgot the glycerol in the enzyme. Your idea is good, and i think it would OK to mix 2 different enzymes together and store them in -20C, is that right?

Ah, wait... Here comes a new idea...
Qiagen have a product called Taq Master PCR mix, that one contains enzyme, buffer and even DNA dye for electrophoresis. And it is 1X concentration, in other word, it's ready to use. I don't think that one contains glycerol. So maybe I should try mix all of them together some time. Of course, not much...

-lee_shore-

Well, like I said, give it a go in a small trial and tell us what happens. If this works, it would be a useful/interesting fact for the community to know about.

However do note Taq is an archea protein and is rather stable. Taq can even survive phenol denaturation.

-perneseblue-

You could also make a stock of your cut plasmid and store it at -20C.

-Andriy-

QUOTE (perneseblue @ Jul 28 2008, 07:50 PM)
Well, like I said, give it a go in a small trial and tell us what happens. If this works, it would be a useful/interesting fact for the community to know about.

However do note Taq is an archea protein and is rather stable. Taq can even survive phenol denaturation.


Ok, i tell you when i do that.

-lee_shore-