How many ligase units? - probably a very stupid question... (Jan/10/2007 )
Hi guys,
I was looking around, comparing ligases from different companies, and I noticed that concentrations are quite different (from 400,000 unit/ml for NEB to 1 unit/microl for invitrogen). I usually use 0.5 ul from the NEB ligase for a 20 ul ligation. So if my calculation is right, that means that I should use 200 ul of the invitrogen ligase, which does not make any sense... What am I missing here???
I was looking around, comparing ligases from different companies, and I noticed that concentrations are quite different (from 400,000 unit/ml for NEB to 1 unit/microl for invitrogen). I usually use 0.5 ul from the NEB ligase for a 20 ul ligation. So if my calculation is right, that means that I should use 200 ul of the invitrogen ligase, which does not make any sense... What am I missing here???
NEB uses different unit definitions than many other companies. NEB= CELU (Cohesive end ligation unit); others use Weiss-units.
1 CELU = 0,015 weiss units
Look at the manual of the supplier which unit he uses and calculate the amout you need (depending on ligase concentration).
There are two radically different units used for T4 ligase "Weiss units" and "Cohesive end units". NEB describes this on their site. The amount of ligase required for sticky end ligations is vastly oversupplied with the 0.5 ul in 20 ul volume you describe. We dilute 20x before using it. For blunt ligations, you need much more, and the "quick ligation" kits often supply even higher concentrations of ligase for this purpose.
I knew that I was missing something... Thank you so much for your help!
One Weiss unit (Weiss B., Jacquemin-Sablon, A., Live, T. R., Fareed, G.C., and Richardson, C.C. 1968 Enzymatic breakage and joining of deoxyribonucleic acid. VI. Further purification and properties of polynucleotide ligase from Escherichia coli infected with bacteriophage T4. J. Biol. Chem. 243:4543-4555.) is the amount of enzyme that catalyzes the exchange of 1 nmole of 32P from inorganic pyrophosphate into Norit absorbable [g,b-32P]ATP in 20 min at 37°C.
One Weiss unit corresponds to 0.2 unit as determined by the exonuclease resistance assay. This definition is rarely used. (Modrich, P. and Lehman, I.R. 1971. Enzymatic characterization of a mutant of Escherichia coli with an altered DNA ligase. Proc. Natl. Acad. Sci. 68: 1002-1005. )
One Weiss unit corresponds to 67 cohesive-end units as defined by New England Biolabs.
In 30 minutes at 16°C, 0.015 Weiss units of T4 DNA ligase should ligate 50% of the fragments derived from 5 mg of lambda DNA digested with Hind III. (J. Sambrook and D. W. Russell, 2001. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Press, Cold Spring Harbor, ME.)
Takara: One unit is the amount of enzyme that ligates more than 90% of 6 µg of λ DNA-Hind III fragments in a 20 µl mixture in 30 minutes at 16°C. One unit of this enzyme corresponds to 0.008 Weiss units by the ATP-PPi exchange reaction in 66 mM Tris-HCl (pH7.6) that contains 6.6 mM MgCl2, 10 mM DTT, 66 µM ATP and 3.3 µM [32P]-Na4P2O7. (Takara website)
Six NEB Units (0.1 Weiss Unit)/20 uL sticky end ligation or 67 NEB Units (1 Weiss Unit)/blunt end ligation reaction is more than sufficient to generate large numbers of colonies. NEB ligase can be diluted to 6 Units/uL and 67 Units/uL in Ligase storage buffer and stored at -20C for one year.