subcloning into a single NotI site - (Jun/08/2005 )

Here is my question: I have a construct which is flanked by both a 5' and 3' NotI site. Is it possible to just digest the construct and the plasmid I wish to clone into with NotI and ligate? If not, how can I go about shuttling the sequence into a different vector? I've tried cloning the fragment with PCR and then ligating, but for whatever reason, it isn't working. I suspect it might have something to do with the fact it is relatively large, but not sure. Either way, was hoping that I could just cut and paste with NotI. So, can anyone help me out here?

hi
i think you can cut your vector and the pcr product both by not I and then paste it in your vector. But maybe you'll have to check the sense of insertion.
you said your fragment was a bit long. What do you mean by long?
how do you purify your pcr product and digest it?

The sequence I'm trying to subclone is about 4kb. I'm actually wanting to cut it out of one vector in which it is flanked by NotI sites and paste it into another vector that has a NotI site (as opposed to using PCR).

Hi,

There shouldn't be a problem with digesting out of one vector with Not1 flanking sites to stick into a Not1 site on another vector. The only thing I'd suggest is to dephosphorylate your vector and try a range of vector to insert ratios when ligating and remember the insert will go in either direction.

Cheers,

Scott

Thanks Scott. I didn't think there should be a problem, but when I tried it at first, I got zero colonies on my plates with insert and something like 3 colonies on my control plate. The vector was CIP treated after digestion, so the only thing I could think of was that maybe I need to change the ratios. In my first attempt, I used a 5:1 (insert:vector) ratio and will just try a wider range and see what happens. I was just wondering if I was missing something more ovbious, and figured I'd check it out.

Thanks for your help.

hi
i had the same problem before, and then realised it was the problem of my ligase. you said you only get three colonies for your control plate. these colonies are more likely to be resulted from the uncut vectors rather than the re-ligated vectors (although after CIP treatment, the chance of religation will be low, you can always gain colonies). so i was thinking maybe there is something wrong with your ligase. i usually add 10 times of ligase that recommended by the company.

Did you puify your vector after CIP txment?

I normally purify after CIP treatment and check DNA concentrations after purification before ligating.

Cheers,

Scott

I also purify and check for DNA concentration after doing the CIP treatment.

As for the cloning, the problem is still not resolved. It looks like that just adding T4 ligase to the insert alone (no vector present) causes the insert to ligate to itself, as I'm going from one band at about 3.7kb (size of linearlized insert) to 2 bands of about 4kb and 2.5kb. When ligase is added to the vector alone, nothing happens at all (at least some control works, right?). So, any further comments or suggestions on this from anyone?

what is your construct that you cut your insert from? how big it is? does it have the same antibiotic resistance gene as the plasmid you try to ligate into? Are you sure that the insert you purified is the one you wanted. it is quite possible that your insert and the left construct is about the same size (as i can see that the size of your insert is quite big).