High frequency of vector religation - (Feb/11/2013 )

I am making a transgenic mouse targetting vector and I am currently trying to insert the 3' ROSA26 homology arm+DTA marker into a KpnI site on the construct.

I am getting tonnes of colonies which I screen by colony PCR and mini-preps and I am just getting a lot of religated vector.

I have tried the following:

A) 1 ug plasmid, digest with 1 ul KpnI-HF with 1 ul CIP for 90 min @ 37 followed by PCR column purification of which 10 ng (based on gel) goes into my ligation.

I first digested for 60 min with KpnI-HF, performed a clean up with a PCR column, then the resulting eluate was incubated with antarctic phosphatase for 60 min at 37, heat inactivated for 5 min at 65 and used 10 ng of this directly in the ligation.

In both cases the insert was digested for 60 min with KpnI-HF, cleaned up with a PCR column prior to ligation. I am using a 2:1 molar ratio of insert to plasmid.

In both cases I am just getting hundreds of religated plasmids. Both my CIP and antarctic phosphatase are fairly new from NEB.

I understand that 3' overhangs are more difficult to dephosphorylate but I really want to crack this problem and finish up the construct.

Any tips for:
-How to reduce the self-religation problem? Using 2 different enzymes for cloning doesn't seem to be an option right now as I'm running out of unique enzymes?
-Any tips to improve success with ligation of large fragments?

Thanks in advance...

You can use PCR to introduce novel restriction sites into the PCR product. You don't need to be restricted to the sites available on the vector.

I would do what phage suggested. Using one RE site to introduce a insert causes more problems than it is ever worth. Even if you get colonies that appear to contain your insert, correct direction of your insert will always be a problem. Take some time, design primers that will give you novel RE sites and skip the weeks of frustration.

One quick point. When I did single RE site ligations, I would always proceed promptly to deposphorylation following enzyme digestion. By cleaning up your reaction mixture, you are giving the DNA ample time to re-ligate. Either way, just put new RE sites in your plasmid.

Thanks for the advice... I hadn't thought the vector could religate during cleanup without ligase?

Anyhow... The vector is 9 kb and the insert is 6 kb. There are very few restriction enzymes left that don't cut either one or the other but I'll definitely re-examine my strategy to see if I can't somehow make this a directional subclone and re-do the PCR with different restriction enzyme sites.

Self-ligation is not favorable, but it can occur depending on the overhang your RE produces and the efficiency of your enzyme. The good thing is all (pretty sure) neb HF enzymes are used in NEB Buffer #4, which (i believe) is the most suitable for CIAP. Double check all that. Just do your digestion (make sure KpnI-HF does not like to remain on ends, if so heat inactivate) and add your CIAP. Again, I would produce novel cloning sites though. The above is alot of work.

A few basic things:

Are you absolutely sure that the Kpn1 is working properly?

If so, check and re-check that the ends you have on the insert primers are the correct ones (primer design mistakes are very easy to make). It is best to have at least 6 bp outside the cut site

Also try some different ratios of insert: backbone, the more insert there is, the more likely it will be inserted.

This might be a silly question but when you say "followed by PCR column purification", do you run a gel and cut the digested vector, or just apply your restriction digestion mix to the PCR clean up kit?

Also agree with Bob1, do you know for sure your enzyme works?

bob1 on Tue Feb 12 03:06:05 2013 said:

Yes I double checked that. The enzyme site on the primers are correct and I have 6 bp outside the cut site.

almost a doctor on Tue Feb 12 13:32:37 2013 said:

I am applying it directly to the PCR column (following dilution with the kit's binding buffer) because there is no fragment released with a single enzyme cut. I find that gel purification in general lowers ligation efficiency because of the UV light damage to the DNA. I only do this if I have to purify the vector away from a released fragment. I do run 2 ul on a gel after purification to ensure it is fully linearized as well as for quantification purposes.

jerryshelly1 on Tue Feb 12 02:55:36 2013 said:

I think you're right. I may try one more time but use Acc65I, which cuts at the same sequence, can be heat inactivated (unlike Kpn) but leaves a 5' overhang which should dephosphorylate more efficiently. In the meantime I'll see if I can't find a way to make this a 2 enzyme subcloning and save myself some grief.

I just want to check that you are cleaning up the PCR reaction prior to cutting with the enzyme. If you don't do this, then the PCR enzyme +dNTPs will extend the DNA and trash the cut end of your insert.