Cloning Problems

Markus Winter via methods%40net.bio.net (by beyonder from blueyonder.co.uk)
Mon Jul 9 05:02:40 EST 2007


On Jun 29, 10:16 pm, Grace Colletti <goldenflo... from yahoo.com> wrote:
> I am trying to clone a 3.2 insert into a 5.3 kb vector (pTRE2hyg Clontech).  I've linearized my vector by cutting the MCS with BamHI & NheI.  I cut out my insert with BamHI and XbaI.  I've tried both CIPing the vector and not (since BamHI and NheI don't create compatible overhangs, I don't think CIPing is necessary??).  I ligate my gel purified products using T4 ligase.  I've tried ligating for 10 minutes, 20 minutes, and 1 hr.  I've done this several times and still I only get vector without insert colonies...does anyone have any suggestions?

Did you do the single enzyme controls? One of them might not cut.

Are you sure XbaI is cutting?  Overlapping dam methylation prevents
cleavage!

How do you purify your DNA? Sugars from the agarose can inhibit
ligation.

Markus




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