Cloning Problems

[Grace Colletti]

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?

 
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