poor ligation of plasmid/oligo construct - (Apr/04/2005 )

Hi,

Following insertion of a 40mer oligo into plasmid, I cut with a unique restriction site within the oligo. I am then trying to ligate this back together again however my ligation efficiency is poor.

I have optimised ligation conditions on the straight plasmid without the insert and it works fine. Does anyone have any ideas why my ligation efficency is decreased when my oligo is inserted?

ok, i assume that you have no problems with the ligation as you were able to recover recircularized plasmids.

what is your enzyme? how long was the digest? certain enzymes actually degrade religation efficiency if overdigested.

that's my first guess, but it could be other things.

keep me posted.

The initial restriction enzyme used is SmaI at 25oC for 30 minutes for insertion of the oligo. This stage rejoins no problems.

The second restriction site following insertion of the oligo is unique to the oligo and is BglII at 37oC for 30 minutes. At this point ligation is poor.

ok, tell me if I'm right with how I understand your experiment.

1) You cut your vector w/ SmaI. Blunt-ligated your 40mer oligo.

- did you do bacterial transformation after this?
- blunt ligation is non-directional, is it important for you to know which way the oligo inserted?

2) you got your vector with the oligo (miniprerp from bacterial clones, I'm assuming). Now all you want to do is to check whether your 40mer is actually in there. So you do a Bgl II digest.

- you see linearization in your gel, voila, you have your insert. although I, myself, wouldve done a double digest with another unique RE as linearization is sometimes hard to judge with smaller plasmids.

- why do you need to religate it? don't you have your original miniprep?

Yep - I cut with SmaI but don't do bacterial transformation and the direction of the insert does not matter. I then cut the construct with BglII which is unique to the oligo insert.

I am trying to assess the ability of different enzyme complexes to ligate the construct back together. However, when using T4 DNA Ligase as a control (which should be very efficient regardless of any complex), my ligation efficiency is significantly lower than when ligating linear plasmid without the construct.

My problem is trying to figure out why??!!

I see what you're trying to figure out.

The only difference I see is Sma I is blunt and Bgl II creates an overhang, but from what you said you saw, then everything seems reversed.

Sorry I can't be of more help.

I have a similar problem..

i cut my vector with 2 different enzyme sequencially (AgeI and SpeI), The anneal my 20 bp oligo (with the 2 overhang on each side).
The ligation is poor, and my plate are full of small colonies that subsequenly don't gro on liquid LB (ampicillin)...

Any idea why?

I don't cip treaty the vector obviously, so i save also the 5' phosphate...

Thanks...

Sbu-
I would check the concentration of oligos you are using, try to make sure that you are in the area of a molar ratio of 1 vector to 3 insert. For a 3 kb vector, 100 ng in the ligation, you would need .6-1.8 ng of insert or 0.1-0.3 pmol of your oligo. Depending on how you are annealing oligo, this could be a 1:1000 dilution or more of the annealed oligos. Using more than this leads to strange results in my experience. Also, make sure that all the solutions that you are using to anneal your oligos and dilute them are sterile.

Thank scotty...

i'm trying with a 1:100 dilution (1micromolar). I use 1 microliter of this with aprrox 10-50 ng of vector....in a 10 microl reaction...

let's see if i will disqualify my boss once in a while...