can I take some of ligation mix and digest? - (Sep/29/2006 )

this is simple. i have ligated the vector to itself so now i want to check it by RE digest. can I simply take let's say 1ul and digest in 10ul digestion mix? won;t the ligation buffer and so on interfere? 1/10 dilution is it enough? please dont tell me i have to ethanol precipitate... thanx for the answers.

-Kathy-

Heat kill the ligase.
Personally i would digest in 20ul, mainly because I don't like working with 10ul. (evaporation problems with the setup I have at my lab, makes the digest rather unstable.)
Unless, the restriction enzyme in use is very picky with its buffer, most will take a little suboptimal conditions. Give the enzyme more time to cut.
Remember to run a raw sample for comparison.

And just out of curiosity, does the ligated vector become resistent to the restriction enzyme, and that is how the vector is being tested?

-perneseblue-

I would worry abt detecting the DNA (insert) on the gel as u r starting with a low amounts of DNA.

-scolix-

Also, I wonder about any possible effect if the vector has been phosphatased? Will the restriction enzyme work on essentially nicked DNA?

-swanny-

thanx a lot guys, well scolix was right. i didnt get any DNA on the gel.... but i electoporated and got some colonies.... so now im minipreping them, but they are so slowly multiplying, this can;t mean that the plasmid is not there is it?

-Kathy-