killer digest issues - (Sep/23/2008 )

Hello everyone!

Well so I heard a postdoc say that sometimes when you do a killer digest after ligation that it stops the transformation from working?

I dont understand why it would do that.....I mean if I have used 50ng of vector for my ligation, in 20 microliters endvolume, and take half of that and killer digest for half an hour with like 1 unit of enzyme I would only be getting rid of false positives. Besides, we transform like 20 or 40 ng of vector into 50 microliters of E.Coli, ive read that some people transform much less than that.

Very confusing, I guess i will try everything out once hehe

Could you explain what is a killer digest? I am not sure what it is?

well for example if upon succesful ligation of an insert with say Bgl2/Xho1 you digest your construct after ligation with an enzyme that is between those sites in your mcs, then you will linearize all of the vectors that religated because that recognition site should not be there anymore if you double digested your vector and cloned the insert into it.

Hmm... This is called a "kill digest" over at my lab.

1 - a kill digest is an extra step, you can lose your DNA when ethanol precipitating the digested ligation mix prior to transformation.

2 - if you transform your ligation mix directed from the digestion mix (without EtOH precipitating and,washing) you will take up some of the digestion buffer. Some restriction digest buffers contain contain detergents, and detergents are seriously harmful to competent cells.

3- ......

hmm.... that is all I can think off right now

haha i think thats what it is called but to make it sound more dramatic i turned it into "killer digest" or "terminator digest" hehehe.

uhm if i start out with 50ng and ethanol precipitate my DNA i certainly could loose it. Uhm could u tell me what protocol u use for this please? I could always take half of the ligation mix and try this out and then transform the other half, see what happens.

Why are detergents harmful to competent cells though? what kind do you mean??

Thanks for the info. We do not do the killer digest for our ligations.

I think after the digest if you can inactivate the enzyme before transformation, then it should be fine

I read somewhere that if you dont inactivate the ligase it sticks to the DNA and inhibits transformation, yet if you have PEG in your ligation and you heat it this also might lead to inhibition.

I will try inactivation I think, you gotta love molecular biology man

I did not heat inactivate the enzyme. However you can try it. I dont think it is an "all or none" critical step.

Yes, do not apply heat (eg heat inactivation) to the mixture with PEG.

we transform the ligation mixture directly without inactivating the ligase.