Weird transformation products - (Feb/08/2007 )

Hi all!

This is my first post on this forum, and I hope it won't be the last !

Anyway, I have some problems cloning a 2,6 kb insert into a 11 kb vector... and since I can't quite do it, I refer to you! So here are the steps I do:

The insert comes from another vector we have. Since I have to add new restriction sites, I PCR amplify the insert with the new restriction sites. Someone already told me that PCR amplified DNA with restriction sites near the ends are quite difficult to digest, so I cloned my insert into a pDRIVE vector (Qiagen), transform into DH5 alpha and purify the DNA with the Qiagen miniprep kit.

After digestion with the two enzymes (SwaI and PacI), I can clearly see my 2,6 kb insert. I then purify it from the gel with an Amicon Ultrafree-DA kit and clone it into my previously SwaI-PacI digested vector (pWPI). After transformation into DH5alpha, I have a rather good amount of colonies. The problem comes when I recover the DNA from those bacteria. The band patterns are really weird on gel, ie do not look like they should when I do my control digestion. I have a major band at 2kb that shifts to 6 kb after digestion (and I shouldn't get this band at all...).

I use T4 DNA ligase for my ligations, and a vector:insert ratio of 1:3 or 1:7. The ligation time vary from 1h at 37oC or O/N at 16oC.

What could possibly go wrong????

Thanks for helping me!

(and sorry for any mistakes in the writing... I'm french )

37oC is quite high for ligation. you may try 1h RT or O/N +4. The ligase buffer might be gone. It cannot stand a lot of freeze-thaw cycles. you may add ATP to your ligation reactions. You may do the ligation reaction in small volumes like 10microliters.
Is there a chance that those colonies you get are self-ligated plasmids? if this is the case, your vector might not be cut properly. How did you prepared your vector? Did you do a double digest or sequencial digest? your insert preparation seems ok.

I needed a sequencial digestion because the two enzymes I use are not combatible (different buffers). Anyway, my empty vector comes from a maxiprep and seems okay (it can be digested with both SwaI and PacI). I doubt that the colonies I get from my ligation are self ligated plasmids because the band pattern when digested with XhoI is different than the one I get with my empty vector. My ligations are all done in 10 microliters.

It is true though that the ligase buffer has been freeze-thawed many times. I'll try to add ATP to it. I'll also try to ligate at a lesser temperature.

Thanks a lot for the quick response dodosko!

I agree with dodosko that 37oC is too high for ligation, room temperature will do.
Probably you may want to check your T4 ligase activity to make sure it works. You can try to religate your digested vector with the ligase.

Yea.. ligation is way too high. Perhaps can try overnight ligation at 4Celcius. Overnight ligation usually works for me.

I am giving my opinions here. Might be wrong though. (I am still very new)

Since you were saying that you obtained a 6kb product, my suspect is formation of concatamers. Few digested vector self ligated or perhaps ligate with your gene of interest.

Perhaps you can try out direct PCR to check the amplified fragment. Hope it helps.

Thanks for the tip guys!

I didn't think that 37C was too high for ligation. I'll try with RT.

I have also been told that the strain I'm using maybe weird also. I'll try with an "approved" batch.