SHIPPING

Shipped in green ice.

FORM

Liquid

SHELF LIFE

12 months

STORAGE

Store at -20°C

UNIT DEFINITION

5,9 Weiss UNITS of T4 Ligase is the amount of the enzyme required to catalyze the ligation of greater than 95% of 1μg of λ/HindIII fragments at 16°C in 20 min.

CONCENTRATION

2.5 Weiss units/μl (500 CE units/μl)

BUFFER

Standard Ligation Buffer, 10x conc.
500 mM Tris-HCl pH 7.8 at 25 °C, 100 mM MgCl2, 100 mM DTT, 10 mM ATP and 25 μg/ml BSA

Quick Ligation Buffer, 2x conc.
60 mM Tris-HCl pH 7.8 at 25 °C, 20 mM MgCl2, 20 mM DTT, 2 mM ATP and 10 % PEG

A white precipitate in the Ligation Buffer is normal and does not affect the reaction efficiency. Do not heat the buffer, as this will damage the contained ATP.

Heat inactivation:

T4 DNA Ligase can be inactivated by incubation at 65 °C for 10 minutes.

Note:

• One Cohesive-End Ligation Unit (CEU) is defined as the amount of enzyme required to give 50 % ligation of Hind III fragments of λ DNA (5’ DNA termini concentration of 0.12 μM, 300 μg/ml) in a total reaction volume of 20 μl in 30 minutes at 16 °C in 1x T4 DNA Ligase Reaction Buffer.

• One Weiss unit is equivalent to approx. 200 CE units.

• T4 DNA Ligase is strongly inhibited by NaCl or KCl if the concentration exceeds 200 mM.

• Ligation of blunt-ended and single-base pair overhang fragments requires about 50 times as much enzyme to achieve the same extent of ligation as cohesive-end DNA fragments. Blunt- end ligation may be enhanced by addition of PEG 4000 (10 % w/v final concentration) or hexamine chloride, or by reducing the ATP concentration to 50 μM.

• To dilute T4 DNA Ligase for subsequent storage at -20 °C a stor- age buffer containing 50 % glycerol should be used, to dilute Ligase for immediate use, 1x Reaction Buffer is recommended.

10X Standard buffer

Vector/Insert DNA

2 ul

100ng-1ug

2X Quick buffer

Vector/Insert DNA

10 ul

100ng-1ug