10 X Ligation Buffer:
666mM Tris HCl pH 7.4
Store at -20oC in small aliquots. Do not freeze-thaw more than 3 times.
For most purposes, it is best to use a ratio of insert : vector >1, e.g.
2/3mol insert : 1 mol vector.
N.B. - you must correct for the relative sizes of the two molecules.
This gives a reasonable number of recombinants without many multiple
For the most efficient utilization of insert DNA, use the following
Fragment Size Vector Concentration
<1kbp 50-100 microgram/ml
<5kbp 25-50 microgram/ml
<10kbp 10-25 microgram/ml
>10kbp 10 microgram/ml
Use excess ligase - but don't go crazy. Diluting the enzyme 20-fold in
the final ligation mixture (final [50u/ml]) works well for all ligations.
Sticky end ligations can be performed at room temperature or even at 37oC
for 2-3h, but work best at lower temperatures. Blunt-end ligations must
be performed at low temperature.
Ligate at 4oC (in refrigerator) at least 6h, best o/n.
Dr Alan J. Cann PhD, Department of Microbiology & Immunology,
University of Leicester, P.O. Box 138, Medical Sciences Building,
University Road, Leicester LE1 9HN, UK.
Email: nna at le.ac.ukhttp://www-micro.msb.le.ac.uk/AJC/nna.html