Purification between digestion and dephosphorylation?

Question

I need to digest a vector (single digestion, sticky ends) and dephosphorylate before ligation with the insert and then transformation into E.coli. The problem is I don't have much of the vector to begin with (v. low yield) and I keep losing alot of DNA in purification. Do I absolutely need to purify between digestion and dephosphorylation? And do I really need to purify again before ligation? I've heard it's not neccessary, but what about the mix of buffers you get? I'll heat inactivate the RE, but do I need to do this with the AP? Any suggestions would be appreciated.

There are no clues in the manufacturers instructions (Roche). I'm using CAP. Anyone know if this is OK buffer wise?

Also I kind of need to not run it on a gel, the plasmid is too large to put through a gel purification column, and I lose too much in purification.

Answer 1

Ok, what I normally do is to digest the plasmid with RE, digesting as much as possible for long time (even over-night). Then, without any purification I treat my digestion with CAP (calf alkaline phosphatase) for one hour at 37C. This phosphatase is generally versatile and works in any buffer, but you might want to check with the manufacturer's instructions for buffer compatibility.
After de-phosphorylation, I make a 1% agarose in TAE with a comb with big wells. If you do not have one, you can tape 2 "teeth" together to make a bigger well. Make sure you add Ethidium Bromide to the gel. I run all my digest on the gel for 1 hour or so at medium volt (so that the gel does not become hot) and then cut the band and purify with a gel purification kit (Qiagen has a very good one). The kit has columns that bind DNA from which your plasmid can be eluted - If you don't have a lot, elute with a small volume of H2O (DNAse-free!).

The advantage of running the gel and purifying the band is that you get rid of all un-digested plasmid which would be useless in the ligation but that would definitely be transformed into your E.coli (quite well, as well!).
Also, if you are cutting and purifying an insert from a different plasmid, you can run vector and insert side by side to see in which proportion to organise your ligation. If you don't have a lot material, try "Rapid Ligation Kit" by Roche. It works at room temperature within 1 hour.

Hope this helps!
Good luck

Answer 2

I usually never purify between digestion and dephosphorylation.....calf intestinal phosphatase usually works well...

good luck