ligation formula

A.J.Cann nna at le.ac.uk
Tue Mar 5 07:36:32 EST 1996


Ligations:
10 X Ligation Buffer:
666mM Tris HCl pH 7.4
100mM MgCl2
10mM rATP
10mM DTT

Store at -20oC in small aliquots.   Do not freeze-thaw more than 3 times.

Planning ligations:
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 
insertions.
For the most efficient utilization of insert DNA, use the following 
guidelines:

	Fragment Size				Vector Concentration				
	 <1kbp						       50-100 microgram/ml
	 <5kbp						       25-50 microgram/ml
	 <10kbp					       10-25 microgram/ml
	 >10kbp					       10 microgram/ml

Enzyme:
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.

Temperature:
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.uk     http://www-micro.msb.le.ac.uk/AJC/nna.html





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