ligation problems - ligation problems (Apr/15/2005 )

Dear all,

I'm completely usuccessful in ligating a NcoI/XhoI fragment into a modified pBluescript vector digested with the same enzymes.
Can anyone help me please?

I'm absolutely sure that the digestion is ok because I have sequenced both vector and insert. I've tried several vector:insert molar ratios, I've also tried the ligation reaction with and without dephosphorilated vector. Everything has been unsuccesful because all clones I obtain contain only religated vector.

I don't know what else I can do. I'm desperate!!

Thank you very much for your help. Best wishes

Firstly, getting colonies back due to religation of the vector means that your digest is incomplete because Xho I and Nco I ends are incompatible and the only way you can get recircularizing plasmids is when you've only managed to cut with just one enzyme.

Incompatible ends usually do not require dephosphorylating.

So, I would work on making sure that your vector is completely digested, indeed, by both enzymes. Now, it's difficult to check for that using gels if the two enzymes are close to each other. Just make sure you let the digest as long as safely possible (to not affect religation efficiency).

Good luck.

Thank you very much for your suggestion but I'm afraid I'm still lost.
I've sequenced the linearized vector digested with both enzymes in order to confirm that the digestion is complete and that the cohesive ends are still in use.
So I don't understand how does the recircularization occurs but it happens. Control plates containing only vector (without insert) result only into one or two colonies. In contrast, 30-50 colonies are grown onto ligation plates. I don't know either how to explain this difference in number of cfu. When I screen all these colonies (either by PCR or by miniprep and restriction analysis) I only find vector.

Well, I'll keep on trying anyway.

Thank you very much again for your help

did you gel purify your NcoI/XhoI fragment prior to ligation?

The only other reason I can think of is that two vectors are ligating with each other, but that hasn't happened to me.

i dunno...

Yes, I used QIAquick Gel Extraction kit (Qiagen). From your experience should I use other purification method?

In case two vectors were ligating with each other I would improve that situation increasing the insert:vector ratio, wouldn't I?.
I've tried ratios up to 50:1 and the result has always been the same.

Thanks for your help

you are right about the molar ratios, and I've used the Qiagen gel extraction kit and it has worked flawlessly for me.

how are you testing for the presence of the fragment? How big is it?

Looking at all these, getting a 20-30X increase in CFU vs. control can only mean that your ligation worked.

The insert is 2.8 kb long and the vector 3 kb.

The screening is based on PCR directly from the colony. Forward primer hybridizes on the vector and reverse primer on the insert. Sometimes I also purify the plasmid and perform a restriction analysis using enzymes that cut in the vector or in the insert in order to check sizes.

I am completely stumped with this.

I guess the last thing to do is to sequence it and see if it is really there or not.

when you do your single digest on the vector, you get a band that corresponds to just the vector size alone?

I'm out of ideas.