PCR & DNA modifying enzymes

Description

Product Details

T4 DNA ligase is purified from E. coli lambda lysogen NM 989.

Reagents supplied: 10x T4 Ligase buffer (w/o ATP)

Unit definition: One Cohesive-End-Unit (CEU) is defined as the amount of enzyme required to give 50% ligation of 6μg lambda DNA-Hind III fragments in a total volume of 20μl in 30 min at 16°C.

T4 DNA Ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5′-phosphate and 3′-hydroxyl termini in duplex DNA or RNA.

Ligation Guidelines:

• Sticky-end ligation reaction:

Components	20μl assay	Final Concentration
10x T4 Ligase buffer	2μl	1x
10mM ATP	2μl	1mM
Linear DNA vector	Variable	50-100ng
DNA insert	variable	1:1-1:5 vector: insert
T4 DNA Ligase (400u/μl)	1 μl	400u
Sterile ultrapure water	Up to 20 μl
Incubate overnight at 16oC or for 30 min at 25oC

• Blunt-end ligation reaction:

Components	20μl assay	Final Concentration
10x T4 Ligase buffer	2μl	1x
10mM ATP	2μl	1mM
50% w/v PEG 4000	2μl	5%
Linear DNA vector	Variable	50-100ng
DNA insert	Variable	1:1-1:5 vector: insert molar ratio
T4 DNA Ligase (400u/μl)	1 μl	400u
Sterile ultrapure water	Up to 20 μl
Incubate overnight at 16oC or for 2h at 25oC

10x T4 Ligase Buffer: 500 mM Tris-HCl (pH 7.8), 100 mM MgCl₂, 100 mM dithiothreitol

Reaction conditions:

• 1x T4 Ligase Buffer
• 400 units T4 DNA Ligase
• 1 mM ATP
• DNA (recommended DNA concentration 0.1 to 1 μM of 5´ termini)
• Optimal ligation at 16^oC

Heat inactivation: 65^oC for 10 minutes

Storage conditions:

• 50 mM KCl
• 10 mM Tris-HCl (pH 7.4)
• 0.1 mM EDTA
• 1 mM dithiothreitol
• 200 μg/ml BSA
• 50% glycerol
• Store at -20^oC

Quality control: Tested for the absence of endo- and exodeoxyribonucleases, ribonucleases and for the capacity to join cohesive- and blunt-ended DNA fragments. More details concerning quality controls could be found in Certificate of analysis.

Notes:

• One Cohesive-End-Ligation unit is equivalent with 0.015 Weiss 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 or hexamine chloride, or by reducing the ATP concentration to 50 μM.