I ligated an approximately 2kb (restriction digested and purified) insert into a 5.3kb vector (restriction digested and dephosphorylated) using a T4 ligase. I used varying insert:vector ratios (5:1, 1:1, 1:5) and when I transformed the ligation reaction into competent cells to did not get any colonies for any of my ligation reactions.
I also ligated the vector on itself as a control and had about 10 colonies on that plate.
Any suggestions for what could be going on?
How did you ligate them? In the fridge 4 degree for 8 h or Have you try the rapid ligation system for one hour in room temperature? Alternate your method according to your buffer, it may help.
Hello Anna,
The usual suspect in that case is (besides the student !! LOL) the solution of the digested insert, the solution of your digested vector is OK since it yields transformants.
I need to know a bit more ... what do you do with your digestion reaction (yielding the insert) ... I think you're isolating the insert DNA on an agarose gel, right? What do you do to go from the DNA band to the insert solution?
Best reagrds
Pr Philippe Urban
Hi there,
If you dephosphorylate the vector, I guess the cloning is made in a single ER site. The control tells you the dephosphorylation is not perfect as you get transformants but at least it's telling you the vector is OK (by the way have you run the control without ligase in order to evaluate the non digested vector?).
The puzzling fact is that you don't get any clone from your ligation which tends to prove the insert is able to interfere with self ligation event but does not allow recircularization between vector and insert. So my thought is that the insert is not perfectly digested. The reason for that :
partial digestion or overdigestion (partial destruction of generated ends) due to digest conditions (time, enzyme concentration)
site too close from the inserts ends to be fully and efficiently digested.
The first thing I would do is to check RE behavior towards sites near DNA ends.
Did you purify the vector after dephosphorylation? Otherwise the phosphatase could dephosphorylate your insert too, leaving no phosphates for the ligase to use.
The most common causes of failed ligations are 1) old ligase buffer in which the ATP is depleted 2) impurities such as chaotropic salts from gel isolation that inhibit DNA ligase activity and 3) excessive UV exposure that damages the DNA in the vector making it unable to propagate in E. coli. To check if your ligation is working, try running it out on an agarose gel. I also agree with Stephen Jung that if you have carry-over CIP activity, you'll damage your insert, so you need to make sure that you perform a very efficient clean up (note that CIP isn't heat inactivatable).
All that being said, the hassle of troubleshooting restriction enzyme mediated subcloning is difficult to rationalize, given the relative ease and high success rate of Gibson cloning. Especially in a case where your cloning is not directional, you should consider adopting this newer technology.
perhaps you should run the whole procedure with a control insert in parallel just to be sure that there isn't something unusual with your own insert. If your insert is designed to express a coding sequence in E coli (is it the case ?) then it might be that it is toxic for your bacteria (even at very low level of expression such as in inducible expression systems).
I must admit that all the tips previously given are correct.
I must admit that 10 colonies after transformation with the ligated original vector as a control is not a very big amount of colonies. in fact, a hundred transformed cells on a plate are far more common (depending on the dilution of course).
As Misses Garcia Fé suggested, cell competency should be checked, and you may have to make a new batch of competent cells (I had this problem once during my thesis).
Sincerely yours.
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