rafael at corona.med.utah.edu
Tue May 23 22:13:58 EST 1995
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?
Rafael Maldonado | "y es que...
room 6160 Eccles Institute of Human Genetics | habemos gente pa to'"
Department of Human Genetics |
University of Utah |
Salt Lake City, Utah 84112. USA. | Joselito "El Gallo"
Rafael at genetics.med.utah.edu |
Rafael at corona.med.utah.edu |
Tel: 801-581-4429 |
Fax: 801-585-3910 |
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