Ligation Problems

[Rafael Maldonado]

On 24 May 1995, Sam Hodge wrote:

> Dear Reader,
> 	           Our lab has been having difficuties ligating PCR products 
> into vectors. The vector has been fully digested and then gel purified 
> using a glass bead kit. The PCR product has been cut ( or we think it 
> has) with two different enzymes. It is difficult to know if the PCR 
> product has been fully digested as only a few nucleotides have been 
> removed from the PCR product. The cut? PCR product is then gel purified 
> by spinning the gel slice through a filter. The PCR product is then 
> precipitated. The ligations have been set up with vector to PCR ratios 
> between 1:1 and 1:10 . The ligation volume is small (10uL) with standard 
> buffer (Tris-Cl/MgCl), DTT and ATP with 2 units of T4 ligase from 
> Bresatec (South Australia). Ligation was left overnight at 14 C. 5 uL 
> of the ligation mix was then transformed using CaCl competent cells.
> 
> 	    Thanks in Advance
> 
I have some ideas:

Are the enzymes used effective cutting near and edge? Some enzymes are 
very inefficient cutting next the end of the molecule. Check out the NEB 
catalog for a list of efficient enzymes at the edge.

Is it a blunt-end ligation? If it is, may be you need more DNA, more 
enzyme, more temperature in the ligation.

Have you tried a dephosphorilated vector?


> 		   Sam I am! 
> 

Do you like green eggs and ham?


Rafa

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                                             |
Rafael Maldonado                             |  "y es que...	  
room 6160 Eccles Institute of Human Genetics |   habemos gente pa to'"
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