RE cloning a 2.3kb fragment into a 4.7kb vector - (Mar/20/2008 )

I am so frustrated...

My insert of interest is 2.3 kb. It came to me in a vector that had been modified in a lot of mysterious ways by another lab. I tried to cut it out of that one a few times, and finally gave up because it didn't seem to work.

I then used PCR to clone it into a TOPO/XL vector. It worked ok. I got a couple colonies, only one was positive, but that's all I needed -- everything's in the sequence that should be there. Life is good.

I did notice that in the TOPO/XL vector, the bacteria were somewhat slow-growing. I came in the morning, didn't notice colonies, and left the plates in the incubator. Six hours later, there were a few small ones, and like I said, one had the insert in frame in the vector.

I went back to RE cloning to try and put it in a GFP vector. I'm cutting with HindIII and XbaI, neither of which are in my sequence of interest. I grew my clones in STBL2 cells, so the methylation sensitivity of XbaI shouldn't have been an issue. I gel purified. I quantified. I calculated. I ligated with T4 quick ligase (1:1, because my bands were too dilute to do 1:3), then transformed into Top10.

Nothing.

Ok, said I. Well, it's a big ligation product. I've had this problem before. Switching to some lovely XL10 gold E. Coli did the trick then, so I tried it.

Nothing.

My cells are competent, so that's not the issue. I don't have colonies in my vector only control, or in my ligation plates, so I don't think it's a problem with the cutting. Has anyone else had this issue? Or have any idea how to fix it? Tomorrow I was going to re-cut, re-purify, re-quantify, and then try using a T4 ligase (1 hour incubation instead of 5 min).

Could treating my purified fragments with something like SAP/CIP help?

In retrospect... there's a post a few down dealing with a somewhat similar problem. I was blinded by irritation with molecular biology.

I guess I'll try the different ligase, and the alkaline phosphatase and then check back. Sorry for that. If you have any other ideas or suggestions, I'll keep an eye on the thread.

Thanks..

one hint: don't overdo UV when gel-purifying! Damages DNA and mostly at the ends that are small single stranded parts...

Hi, Do not do the CIP on your fragment , do it on your vector and if you can do gel purification of your vector after digestion and CIP. You have to increase your Vector/fragment ratio even if your fragment is diluted just increase your reaction volume. You can also do precipitation to concentrate your fragment.
Hope that going to help.
Good luck.

A 2.3kb insert isn't all that large. I subclone with inserts that large (and much larger) all the time. My main concern is the ratio you used. You really need to have the 1:3 if you are having trouble. Sometimes you have to go up to 1:5 or 1:10. If you don't have much insert, just use less vector. Ligations can be inhibited by too much DNA and are usually much better with minute amounts of DNA. Definately CIP treat the vector (not your insert). This takes away the phosphates on the edges of the vector so it can't re-ligate. You should see fewer empty vector colonies. By the way, I always have slow-growing colonies with the TOPO vectors. The only other thing is, make sure you are growing a full hour before plating and you may need to let your plates incubate for two days. A post-doc in my lab tried cloning for months until he accidently forgot his plates for a day. Two days later there were colonies (still pretty small) which had his insert!