Xba I and subcloning - (Jan/23/2006 )

Hi All,

I am facing a strange problem with subcloning these days. I have a plasmid DNA which is ~5.5kb and is not methylated. XbaI linearizes this plasmid. I am trying to subclone a ~4kb fragment also cut with XbaI (I am not sure if this DNA comes from the plasmid which is not methylated, but is a part of pGEM 7 vector), but is cutting with XbaI properly. The problem is that when I am trying to ligate plasmid DNA cut with XbaI (CIPed) and gel purified with 4kb band cut with XbaI and gel purified-- I am not seeing if ligase is working at all. I usually run 5ul of sample after ligation on gel to see if ligation has worked or not. I changed the ligase enzyme twice and changed the incubation time from 2 hours to overnite at room temp., still the problem persists. Anyways I proceeded for the transformation. I did get some colonies on the plate, but none had the insert.

What should I do? I am guessing that this is something to do with methylation and XbaI. But whatever I read about XbaI, it will not cut if the DNA is methylated. In my case XbaI is cutting properly. The only problem I am facing is the ligation.

Can anybody help me out with this problem. Any help is greatly appreciated.

Thanks

Use a different restiction enzyme if at all possible.

Here it is again gang. Another ligation problem with Xba.

Question: You are doing a single enzyme digest? So you are trying to stick an insert with 2 Xba sites into a vector linearized with the same? If so have you dephosphorylated your vector after digesting it? If you don't there is a good probablility of the vector simply self-ligating and leaving your insert out in the majority of the ligation events, esp with a larger insert.

This may not make sense (don't know how long ligase usually lasts in a RT reaction), but maybe you should ligate at 4C O/N instead of at RT, perhaps the ligase is not working after only a couple of hours at RT so it really isn't helping to go overnight??

I recall pBluescript discussing problems with both XhoI and XbaI in an older thread. More recently, this thread and another discussed problems with Xba and Xho independently. This is funny, actually, as I tried to use XhoI and XbaI for a ligation... I struggled for 2 months wtihout success!

Strange and interesting.

-Hank

perhaps try using Shrimp alkaline phosphatase to dephosphorylate. I don't know if this is the problem but if you are getting a bunch of native vectors it could be. Shrimp phosphatase is much easier to use and you can go straight from dephosphorylation to ligation after heat inactivation without purification steps and other inactivation steps like you do with CIAP.