Ligation problems

Ke-Xue Huang khuang at BIOC.RICE.EDU
Fri Jul 31 07:54:59 EST 1998


Hi, York,

I think BstxI is a real problem(but I dont't know why), we tried to put 
BglII and BstxI fragment to BamHI, BstxI digested vector, it never works. 
Of course, we digested the vector and insert seperately and gel purified it.
Try another way!


Kexue

On 28 Jul 
1998, Ian A. York wrote:

> 
> We're having a terrible time getting what should be a straightforward
> ligation to work.  I have a feeling we neglecting something obvious, so I
> won't be offended at simple suggestions ...
> 
> We have purified some genomic DNA and we want to put it into an expression
> vector.  We have sheared it, blunted, ligated BstXI adaptors, and ligated
> into complementary BstXI sites (Invitrogen vector, Invitrogen adaptors).
> Result--very inefficient ligation.
> 
> Just blunting and ligating into dephosphorylated blunted
> vector--inefficient.
> 
> We tried using very degenerate primers and PCRing a smear up (there was a
> reason for this, too complex to explain now) and tried using TA cloning to
> put into the vector (home-made TA cloning system).  Inefficient.
> 
> Tried blunting the PCR product and ligating into dephosphorylated
> vector--inefficient. Blunting and BstXI adaptors/BstXI-cut
> vector--inefficient.  For the PCR products, we tried using the proteinase
> K treatment protocol to eliminate residual polymerase--no help. 
> 
> We use NEB's T4 ligase, standard conditions as they recommend.  We can run
> gels on the ligation and we do not see evidence of ligation there, so it
> probably isn't a question of inefficient transformation.  We've used
> appropriate competent bugs (XL-1 from Statagene); test transformations
> give acceptable results



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