Problem with Digestion or Ligation. How to tell? - (Apr/14/2007 )

I'm trying to insert 500 bp into 4.5kb vector. It hasn't been working so I tried to put in as many controls as I could. I'm double digesting the insert and vector with NotI/EcoRI. I also single digested the vector with EcoRI. I PCR purified the insert, gel purified the vectors. I ligated with T4 DNA ligase the insert+vector and just the single cut vector. I also ligated the single cut vector in the "old" ligase buffer that I had been using and also in the "new" unopened ligase buffer. Then I transformed my insert+vector, the "old" buffer religated vector and the "new" buffer religated vector, and uncut vector. Today was the day. I pulled the plates out of the incubator.... Growth with the uncut vector and that's it.

So it's a ligation or digestion problem, right?? How can I tell? Do I need to try a NotI single cut vector along with the EcoRI. Is there a way I can run the ligation out on a gel to see what's going on? Help please!!!

Yes, there is a way to test ligation. You should take your insert only and ligate it in the presence of EcoRI. Do another ligation in the presence of NotI. Do the ligations with normal T4 ligation buffer (not quick ligase buffer). Heat kill the ligations and REs at 80C for 20 minutes. Run the results on a gel. You should see just the double length fragment. Any single length fragment you see is a failure of the cutting, damage to your cut ends, or failure in the ligation. You can tell which one of the enzymes is failing this way. Do a similar experiment for the vector. The heat killing of the ligase is required to see the DNA on the gel, but it won't work with quick ligase or other PEG containing buffers. You can also do the ligation and digestion separately, which may be easier, and allows you to also run the uncut ligations on a gel. These are a lot harder to interpret, however.

Things to watch out for:
* DNA damage due to UV exposure during gel purification -- use 365 nm UV or blue light and short exposure
* Poor cleanup of PCR enzymes and dNTPs prior to cutting -- the enzymes fill in the cut end
* exonuclease damage to cut ends (store DNA in TE)
* inadequate overhang on NotI PCR fragment cut ends especially

I'm not sure I understand how to test the ligation... Ligate it in the presence of the single enzymes?? What temperature would I run it at? Also it may be problematic cause my insert is polyclonal so I don't get a nice 500 bp band when I run it on a gel, I get a smear instead... So I guess I could still see a double length smear.

I've been digesting my insert with NotI overnight then adding EcoRI for an hour or so at the end. I have 12 bp at the end of the NotI site and the NEB website suggests overnight for 11 bps. And the PCR cleanup is done with a qiagen PCR purification kit. That should be sufficient, hopefully?

Thanks for the help!

I think phage means ligate single-digested insert, ie, EcoRI-digested or NotI-digested insert. That will test for the digestion.
A simple way to test the ligase itself is to try to ligate your DNA ladder (assuming it's not already in loading buffer!!!). You should get a smear of high MW DNA, and the ligation reaction can be as short as 30 min at RT.

unfortunately my ladder's already in loading buffer... but it's a good idea! I'll see if I can scrounge up some that isn't.

Ligate the single digested insert... Ligate it to the double digested vector? Then if it ligates I'll get one larger band. And if it doesn't ligate then there's a problem with the digestion?? Is that what you mean??

Thanks for all this help. I'm pretty clueless here and feel that I'm out of options.

1. Ligate your insert to itself. Do this with normal T4 DNA ligase buffer. 10-30 minutes at room temperature will work. Heat kill the ligase at 80C for 20 minutes. Split this into 3 parts. Cut one with EcoRI. Cut another with NotI. Run a gel with:
* unligated insert
* ligated insert
* ligated insert EcoRI cut
* ligated insert NotI cut

You should see double length bands on the ligated/cut lanes compared to the unligated insert. You should see long fragments in the ligated insert (uncut) lane.

Repeat this with the vector. Note that unless the vector has been gel purified, ligation will sometimes result in reinsertion of the fragment removed.

This will all be much more difficult with a variety of insert fragment lengths. Perhaps you could get the ligations working with a simpler control experiment.

I like this idea a lot! I really appreciate this. I'd rather not have to make more DNA if I could help it, so how much DNA do you think is needed to test this out?? Would 50 ng be too little or too much?? I'd rather get this tested asap if I could.

Thanks!

You need to use as much as it takes to see clearly on a gel.