(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
> 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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