Ligation is killing me - 1kb insert, 6 kb vector (Nov/01/2005 )
I've been cloning fairly regularly for 7 or 8 years, so I more or less know what I'm doing. But lately, I haven't had any success.
I am trying to insert a 1 kb PCR fragment into a 6 kb vector.
All REs are NEB, ligase and buffer are Invitrogen. PCR oligos have 4 extra bases beyond RE site. I have the complete sequence (verified with RE digest) for both. REs being used are Xho and Xba (both Buffer2).
Protocol is basically: PCR - 50uL (check aliquot on gel). Clean up with Qiagen PCR Spin columns.
Digest in 50 uL 2hr at 37 deg C. Digest 5 ug vector in 50 uL. Run all of both on gel, cut bands, extract with Qiaex II, quantify by OD260, and verify by running an aliquot on a gel.
Ligation is 3:1 molar ratio, 50 ng vector in 10 uL. (2 uL 5x buffer, 1 uL T4). RT for 1 hr.
Add to DH5alphas on ice 20 miin, heat shock 42 deg for 45" - 1', on ice 10 min., add 800 uL LB, shake at 37 for 45 min, spin 1 min at 3 krpm resuspend in 120 uL, plate on prewarmed LB-amp plates.
I know the competent cells and all lab reagents are fine and work well, as all my labmates have NO problems using the same reagents. I have 3x sequencing coverage of my insert, so i know the sequence cold. So, what the h*** am I doing wrong? Any ideas?
At this point, I have a backlog of a dozen or so constructs, none of which are working, and this is just one representative.
I tried running a few uL of my latest ligation attempt on a gel, and saw my insert, the insert dimerized with itself, and what appeared to be a double banding of the 6 kb insert, which i assume is the insert dimerizing as well. I did not see any supercoiled. Should I expect to?
Any help/advice/comments/thoughts would be greatly appreciated.
Should work, check that the XbaI site in the vector is not methylated.
No, you shouldn't expect to see supercoiled plasmid after ligation.
I agree about checking XbaI methylation. But you can verify this experimentally by running your vector on a gel. There should be no problem in cutting a PCR product, since it is not methylated.
If I were having problems for more than a few tries, I would TA clone this with the Invitrogen Topo-TA cloning kit, grow the plasmid, and cut the insert from the plasmid.
Something to think about is UV damage to the DNA during the gel isolation. Use long wave UV (365 nm) to visualize, or cut without UV exposure by cutting after visualization of a sacrificial band.
Thanks for the tips. As for Xba methylation - that's not a problem in this vector. It's one my advisor designed as a post-doc from other starndard vectors, and the MCS is specifically tailored to using the Xho-Xba pair. That said, I always carry along single digests of my vectors to ensure no problems, and Xba cuts fine.
As for the TA cloning idea- I actually thought of that about 10 minutes after posting this. That'll be a great way of ensuring that I am actually cutting the PCR product, since you can't really tell that by running the PCR band side by side with the cut PCR. (without long run times on high % gel).
I hadn't thought of the UV damage aspect - that's certainly a possiblity. One other possible snag is that I am adding Qiagen's Q Solution to my PCRs - I'm pretty sure it's just betaine. Qiagen Tech support assures me that the spin columns remove all traces of it and that it shouldn't inhibit my ligations, but as it's the only protocol difference between me and the rest of my lab, it's a stand out target. So, I'm trying a test run of this ligation from a PCR without the Q soln.
Thanks again for all the help.
I'll keep you posted on what winds up working.
Hey Matt, I spent about 3 months trying to do a 1.5kb fragment into a 6.5 kb vector Xho/Xba. Never worked. Did/thought all the things mentioned in this thread. I fixed the problem by changing RE's.
Will never do an Xho/Xba again.
Will never do an Xho/Xba again.
You sure you are giving them enough overhang? 4 is not always enough.
So no Xho/Xba, even with TOPO?
Wow, that's good to know. What is wrong with this interaction?
You sure it's not the methylation with the XbaI?
I did not try TOPO.
I don't know what the interaction is, but this is the third time this incident has come to my attention.
Thanks for all of you ideas and help. I finally got my issues worked out. 9 of the 11 different cloning constructs I've tried in the last 3 days have worked beautifully.
The problems were twofold: First, it looks like Q solution (betaine) probably inhibits NEB's restriction digests. I called Qiagen customer service to find out about it, but they seemed fairly sure that using their PCR spin columns to clean up the PCR was sufficient to eliminate the Q sol'n. But I found that my PCR products were not being cut when I added the Q sol'n to the PCR rxn.
Second was that we make our own competent cells in the lab, and apparently, the last batch of cells was weak enough that low efficiency ligations (like those inhibited by uncut ends thanks to Q sol'n) just didn't get transformed at all. I tried our latest batch of cells (just made this past weekend), and they almost all worked like a charm.
So, word to the wise - only use high competency comp cells, and don't use Q sol'n in PCRs that need to be digested, unless you plan on phenol chloroform extracting them.
Ugh... I suppose this probably explains the last month and a half of failure for me. Thanks for the info!
«PCR oligos have 4 extra bases beyond RE site.»
I think 4 base extra base pairs is not enough.
Check on Biolabs catalog.
Xba I requires 5 extra bases; XhoI, I am not sure but I think is also 5 bases.
You might not have a problem digesting the insert. So if you decide to clone in TOPO might be easier, otherwise just design 1 or 2 base pairs longer oligos