Ligation problem, might be bc/ it's the 5kb insert? - Vector and insert are pretty much same size.. and it's not workin in a ' (Sep/13/2006 )

Hi guys,

I've been wondering around on the site for several hours now and I noticed there are a lot of experienced people here that like to help out unexperienced or very troubled people... I like to make more use of that!
This is my problem. I'm not sure were it exactly is, if it's the transformation (didn't run a plasmid control, I know stupid! For several times.. even more stupid!) or the ligation.
My guess would be the ligation but I definetly might be wrong about that and I'm willing to try pretty much everything!
So here it goes.

My insert is 5 kb (pretty big is what I'm thinking) my vector is 5.6 kb.
They are both cut with NotI and XhoI. First problem is that in the vector these are right next to each other. I've done a digest with NotI first (because it need more overhang) and after 30 min. I've added XhoI (this should work, is what people tell me). Of course I ran both on a gel and they looked to me as they should look. But I obviously I can't see if the DNA is linearized by 1 or 2 cuts.
After gel purification I did a ligation w/ different ratio's vector : insert, 1:3 1:7 and 1:14 and vector only and insert only. I let that sit for an hour and overnight.
Then the transformation where I used 2 ul for each and by now I have done it 3 times and haven't gotten any!!.. Even on my vector only and insert only.
Since this is pretty much my second month in cloning I have no clue what it all could be
And was hoping that some of you could help me out with some ideas to try.
Pretty please?

Thanks a lot in advance.

repeat it, but do the ligation and transformation controls. this is the only way to find the source of the problem

on the surface, it looks OK. perhaps you can cut longer. how close, is close with the two sites in the vector? this could give you a problem

I was following a paper that told me they used overlapping sites (one base overlapped) to clone...may have been the same enzymes? I tried for over 2 months before I scrapped it and went with a different tactic. I don't know if they just got lucky or what, but I think sites that are too close do not cut properly

5kb insert
5.3kb vector.

Those pieces aren't dangerously big, so have no worries. But at these sizes, your vector prefers to self ligate. So keep that in mind.

Well, i guess you have already punished yourself for not running a gel on the ligation mix. Please do run a test gel in future.

Firstly how much DNA are you cutting? Half an hour sound pretty short

(although I cut massives amounts, 15 ug, yes it is wasteful, shame on me. But I like to be wasteful. I subscribe to the brute force and ignorance method.)

As for the Not cut, I you are worried about the cut. The short time bothers me. Run a gel to show the DNA has linearise. Once done, add Xho.

And aimikins has a point. Stating the distance between XhoI and NotI sites would help.

If the end is too close to the site, efficiency drops. I would probably leave the Xho cut longer. Could you state how long thw DNA was cut with Xho?

Was dephosphorylation carried out on the vector. I would recommend it.

Finally there is always the bad ligase and bad ligase buffer senario. Get some new ligase buffer, melt it with your hand, vortex and give it a good sniff. Memorise that smell. New ligase buffer smells strongly of DTT. If that smell fades, your buffer is going. If the smell is gone, so has the buffer.

T4 Ligase is probably the most unstable commercial enzyme I have ever had to use. It doesn't like freeze thawing, and I understand that a former lab collegue had discover the enzyme goes off at minus 20 but is stable at minus 80. This this true? I don't know. But considering how many ligase vials have gone bad in the lab, i wouldn't be surprised. (And we keep the ligase in a cryo cooler... some fancy freezer rack. Ice isn't good enough for that spoiled baby.)

P.S: if you use column purification, make sure that both insert and vector a free from wash solutions. Residue from the wash solution severely interferes with the ligation reaction.

Thanks guys, I definetly have some ideas now. Indeed the controls.. I slammed myself in the head because of that.. But really my other ligations that I did before this went fine, I guess I was just hoping in stead of thinking.

To give you a better picture between the NotI and XhoI sites there are 6 bps. So I'm thinking I want to look for the same vector that already have an insert in there. I was cutting 1.5 ug DNA w/ 2ul of enzyme (both) so that should have been fine. Both enzymes for about 35 min.

Indeed I'm using T4 ligase, I will try a new ligase and a new buffer, never really smelled it.. Not sure what to think about it either!
I did run the gel after the first cut and saw that the DNA was linearized. After the second it still is but of course I don't really see a different in size.