Linear fragment uptake by E.coli - (Apr/14/2007 )

I have a silly problem.
Trying to clone 2kb frag. in 10kb vector. The problem is that my fragment has all the restriction sites that can be used to clone it into vector. So, I tried blunt ligation (using incompatible end enzymes) and wasn’t surprised that it didn’t work.
But while I was ‘fulling’ around with blunt ligation - digested vector with HindIII, filled in the ends with Klenow, dephospho. it, run on the gel, gel purified and with no ligation did transformation of that linear fragment. There are some colonies, but due to time and other things I did not do mini prep of these colonies.

So my question is, is E.coli able to ligate that fragment once it is taken in?
I think I red somewhere that bacteria can do that if the fragment is useful to it, but maybe I am hoping too much.

Thank you

theoretically it can. And I have read a paper where the researcher exploited E.coli dislike for breaks in DNA, and used E coli's DNA repair machinery to ligate the DNA fragments (sticky ends) together and thus obtaining a fully functional plasmid.

Neat trick. However, I have not tried this myself and it looks like a long short. I have not read about anybody else using this marvelous technique, which may indicate it isn't as easy as it would seem in the paper.

My suggestion would be to PCR your fragment, using primers that add unique resistriction sites to the fragment which would allow you to ligate said fragment into your plamid. Do remember to add enough basepair around the restriction site, as all restriction enzyme have a foot print, ie they need space to sit on while they work. NEB's web site has the technical guide on enzyme cutting efficiency close to DNA ends.

Some restriction enzyme produce the same overhangs, thus site cut by two different enzyme may actually produce sticky ends that are ligatable to each other
examples are
BamHI - BclI - BglII

XbaI-SpeI-NheI

XhoI-SalI

Thanks for lifting up my spirits again.

I want this thing so I’ll set up colony PCR on all the colonies for blunt ligation.
Also I was going for same sticky ends (PstI - NsiI) but that is one end only, so for the other I hoped that blunt end (can use blunt end enzyme) would do the job.

The paper you read, if I use something that produces approximately same ends maybe E.coli will smile at me.
Will let you know in few days.

Primers are out of the question, need to do what needs to be done with what I have.