Can't ligate 2 linear fragments with cohesive ends - (Jun/12/2007 )

I am trying to make a chimera between the 5' end of one variant (A) of a gene and the 3' end of another variant (B ). I have both variants cloned into the same vector. This is what I'm doing:

cut inserts (3.2kb) out of vectors using MluI and NotI (the sites they were cloned in with)
clean up reaction using Qiagen PCR purification kit
cut envelopes with MfeI (to generate an 1.8kb and a 1.4kb fragment)
gel-purify MfeI fragments with Qiagen kit
set up 2 ligation reactions: 5'-A + B-3' (8.5 uL each), 2 uL T4 ligase buffer, 1 uL T4 ligase and the same reaction with A-3' and 5'-B
ligate (I have tried 16 degrees ON and benchtop for 1 hour)

When I run this reaction on a gel alongside the same quantities of the MfeI fragments that I used in the ligation, the MfeI fragments look lovely and bright but the ligation reactions consist of very faint smears with a few discrete bands, none of which are at 1.4kb, 1.8bp, 3.2bp or any other reasonable size. The most prominent bands are at 1.5kb and 2.1 kb (they are clearly different sizes from the MfeI fragments), 4.0 and 4.5kb, and 10kb or bigger. I would think the ligase is contaminated with a nuclease or a restriction enzyme or some such except that all the bands are *larger* than they should be. It seems this should be simple, but apparently not for such as me I would really appreciate any insight into what I am doing wrong, I'm not terribly experienced with or good at cloning.

(MfeI cuts the vector itself only once to generate a 170bp fragment [the vector is 5.4kb], but since I gel-purify the 1.4kb and 1.8kb fragment, I would not think this is part of my problem, but I am usually wrong about these things.)

umm... could you please explain this ligation strategy again. I don't quite understand what is going on.
What was the insert cut with? And what was the vector cut with?

I'm not trying to ligate an insert into a vector. I'm trying to ligate two linear fragments together after cutting them with MfeI.

I see. Two fragments, with each fragment being exised by MfeI?

If both fragments have MfeI ends on both ends of the molecule, what you will get is a mixture of molecules in every possible combination and concatemeres in every possible size, with a tendency for longer/larger concatemeres. You will also get circular molecules, where the Mfe ends recircularise. THese circular molecules do not run like like linear counter parts, they move slower, thus appearing larger then they really are.

I am guessing that is what you are seeing. Where is the vector? The vector is required in the multiway ligation, else most of the inserts will be locked up in large concatemeres/or circular molecules.

She's excising two variants of the same gene with MluI and NotI. Then she is cutting each one into two fragments with MfeI. She gel purifies each fragment (4 at this point) and then matches one fragment from the first variant with the other fragment of the second variant, making two different chimeras.

I'm not sure if running your ligation reactions on a gel gives you a clear picture of what's going on. as perneseblue says, try and include the vector (cut w/ MluI and NotI obviously) in the ligation reactions with 5'-A + B-3' and A-3' + 5'-B. Then transform the ligations and plate them so that you can do a miniprep. After you have your miniprep, make several digestions:
1. with MluI and NotI - should give you a 3.2kb band
2. with MfeI - should give you two bands - because it cuts once in your vector and once in your insert right?
3. with MluI and NotI and MfeI - should give you four bands - 170bp band, 1.4kb, 1.8kb, and whatever is left of your vector.

If your analytical digest turns out the way you expect, then you have a winner! But anyway, there are so many steps involved, a lot of things could go wrong so maybe this suggestion might not be practical in the long run. After all, I am no cloning expert.

alex

You can visualize the ligation products easily on a gel. The trick is to heat kill the ligase, and to avoid the "quick ligation" kits. If you use normal T4 DNA ligase buffer, and then heat kill the ligase at 80C for 20 minutes, then you can run the product on a gel and see clear bands. In this case, you should ideally see three bands, the double length of each part and the desired linear fragment. There will likely also be some unligated DNA from each part. Run control lanes of each part, and each part ligated with itself.