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  PCR & DNA modifying enzymes

T4 DNA Ligase

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.

(SKUs: PD006S, PD006L)

60.00144.00
  • 400u/μl
  • 30000U
  • 3x30000U

 

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:
Components20μl assayFinal Concentration
10x T4 Ligase buffer2μl1x
10mM ATP2μl1mM
Linear DNA vectorVariable50-100ng
DNA insertvariable1:1-1:5 vector: insert
T4 DNA Ligase (400u/μl)1 μl400u
Sterile ultrapure waterUp to 20 μl 
Incubate overnight at 16oC or for 30 min at 25oC
  • Blunt-end ligation reaction:
Components20μl assayFinal Concentration
10x T4 Ligase buffer2μl1x
10mM ATP2μl1mM
50% w/v PEG 40002μl5%
Linear DNA vectorVariable50-100ng
DNA insertVariable1:1-1:5 vector: insert molar ratio
T4 DNA Ligase (400u/μl)1 μl400u
Sterile ultrapure waterUp 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 16oC

Heat inactivation: 65oC 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 -20oC

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.

 

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