Cloning Problems

ChenHA via methods%40net.bio.net (by hzhen from freeuk.com)
Sat Jun 30 18:58:08 EST 2007


Grace Colletti 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.  

What is the source of the insert?  If it's a PCR product, did you make 
sure that there are enough extra bases at the end to ensure the enzyme 
can cut?

> 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??).

It is not necessary.

> I ligate my gel purified products using T4 ligase.  I've tried ligating for 
> 10 minutes, 20 minutes, and 1 hr.  

Are you using one of those rapid ligation kits?  If not, the standard 
ligation time is 2 hours at 12-16 degC.

> I've done this several times and still I only get vector without insert colonies...does anyone have any suggestions?
> 

Read the other threads.

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