Efficiency of restriction enzymes

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huyenkobe
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Efficiency of restriction enzymes

Post by huyenkobe » Thu May 12, 2011 8:32 am

I have used some restriction enzymes for my experiments and I found that unlike what is announced at New England Biolabs brochure where affirm that the efficiency can be maintain after the expired date, it really reduces every time I use. For the first time I used AsisI and MluI to clone my insert into plasmid. I screened 28 colonies and got 26 colonies with the expected insert inside. But this rate reduced just at the second time and at the fifth time, I had to screen 48 colonies to get 2 expected bands. But anyway, I got it thank goodness. Now I repeat this experiment and even I screened more than 1000 colonies I could not find any result. The size of the insert and plasmid does not different from previous experiments. What should I do now, should I throw the enzymes away even though they have not been out of date yet. Any suggestions?l

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JackBean
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Post by JackBean » Thu May 12, 2011 8:53 am

That doesn't look like problem of REs to me, but rather the ligation, transformation etc.
Do you have some pure DNA, which should be cut by these enzymes? If so, try it and you will see,whether is it cut or not. If it is, your enzymes are fine, if not, they are dead and you should discard them. But I would bet, they are still fine.

(are you keeping them cold all the time?)
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

huyenkobe
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Post by huyenkobe » Thu May 12, 2011 11:37 am

Dear JackBean,
In my lab, we do not have DNA to check those enzymes and we are in economical trouble now, so my prof does not agree to buy such DNA. I repurified my plasmid several times but I can not get a plasmid with double digestion. Anyway, without pure DNA to check the efficiency of digestion enzymes, do you know the other ways to check their function? thanks for your help.

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