LIGATIONS

DNA Ligations

Preparation of vectors: Digest the vector with the appropriate restriction enzyme and check for comple te cutting on an agarose gel. To prevent self and concatomeric ligation of the vector, treat this DNA with either alkaline phosphatase (BAP or CIAP) to remove 5' phosphates. For BAP treatment, add 50 U per µg of DNA (in restriction enzyme buffer is fine) and incubate for 1 hour at 60-65°C. BAP is most effective on 5' overhanging ends; blunt ended DNA is moderately resistant to BAP treatment, and treatment with BAP is virtually ineffective with 3' overhanging ends (ex. Pst I). Use 200 units per µg of blunt-end DNA.

Plasmid Ligations: For insert sizes of a few kb or less, we typically ligate a 2:1 ratio of insert to vector DNA for plasmids, and between a 3:1 and 5:1 ratio for M13 vectors for overhanging end ligations. For plasmids, we typically ligate 200-300 ng of insert to 100-150 ng vector in a 15ul volume. Blunt end ligations in plasmids are often done with 25 ng vector and 125 ng insert (or scaled up in quantity when larger amounts are needed, i.e. it didn't work the last time). It is preferable though to use about a 2:1 molar ratio of insert to vector. Add the vector and the DNA to be ligated to ligation buffer (diluted from 5x or 10x stocks) and heat for 10 minutes at 65°C. Add 1.5 µl of 0.1 M DTT, 3 µl of a 1:100 dilution of 0.5 M ATP, and 1 unit of ligase. Ligate at 15°C for a minimum of three hours using 1 unit or 3 units T4 DNA ligase for staggered-end or blunt-end ligations, respectively. Use half of this solution for a transformatio n, keeping the other half in case the transformation efficiency is low.

M13 Ligations: We typically ligate 5:1 weight ratio of insert to vector DNA, irrespective of whether or not it is staggered or blunt ended (ex: 40 ng M13 and 200 ng insert). The ratio may be increased slightly (2x) for large inserts > 1kb, but maintaining large inserts in M13 is difficult. Add vector, insert, and ligase buffer and then heat to 65°C for 10 minutes. Add DTT as above and 1/10 volume of a 1/5 dilution of 0.1 M ATP (final [ATP] is 2 mM) to ligase buffer. Add 1 unit of T4 DNA ligase for staggered end ligations or 3 units for blunt end ligations.

RECIPES:

5x Ligation Buffer (250mM Tris pH 7.6 and 50mM MgCl2): 0.5 ml 0.5 M Tris-HCl pH 7.6; 50 µl 1.0 M MgCl2; 450 µl autoclaved water; - or - use ligation buffer supplied by enzyme manufacturer.

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Copyright 1993, 1996, 1997, and 1998 by Mark Barton Frank, Ph.D.
Proper citation for data acquired from this document is: "Frank, M. B. DNA Ligations. In: Frank, M. B. ed. Molecular Biology Protocols. (http://omrf.ouhsc.edu/~frank/ligation.html). 1997. Oklahoma City. Revision Date: July 28, 1998."