T4 DNA Ligase

E. coli lambda lysogen NM 989

Cat. No.	Amount	Price (EUR)	Buy / Note
EN-149S	400 Weiss units (80000 CE units)	37,60	Add to Basket/Quote Add to Notepad
EN-149L	5 x 400 Weiss units (5 x 80000 CE units)	149,70	Add to Basket/Quote Add to Notepad

For general laboratory use.

Unit Definition: One Weiss unit is defined as the amount of enzyme required to catalyze the exchange of 1 nmol of ³²P from pyrophosphate to ATP, into Norit-adsorbable material in 20 minutes at 37 °C.

Shipping: shipped on gel packs

Storage Conditions: store at -20 °C
avoid freeze/thaw cycles

Shelf Life: 12 months

Form: liquid (Supplied in 10 mM Tris-HCl pH 7.4, 50 mM KCl, 0.1 mM EDTA, 1 mM DTT, 200 μg/ml BSA and 50 % [v/v] glycerol)

Concentration: 2.5 Weiss units/μl (500 CE units/μl)

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

Content:
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

Fast 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

component	EN-149S	EN-149L
T4 DNA Ligase	160 μl	5 x 160 μl
Standard Ligation Buffer, 10x conc.	1 ml	5 x 1 ml
Fast Ligation Buffer, 2x conc.	5 ml	5 x 5 ml

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 storage buffer containing 50 % glycerol should be used, to dilute Ligase for immediate use, 1x Reaction Buffer is recommended.

Assay Set-Up:
Standard Ligation Assay

comp.	final amount/conc.	20 μl assay
Standard Ligation Buffer, 10x conc.	1x	2 μl
Vector/Insert DNA	100 ng - 1 μg	100 ng - 1 μg
T4 DNA Ligase	0.1 - 1 Weiss units	0.04-0.4 μl
PCR-grade Water	-	fill up to 20 μl

Incubate for 20 - 30 min at 16 °C for optimal ligation.

Fast Ligation Assay

comp.	final amount/conc.	20 μl assay
Fast Ligation Buffer, 2x conc.	1x	10 μl
Vector/Insert DNA	100 ng - 1 μg	100 ng - 1 μg
T4 DNA Ligase	0.1 - 1 Weiss units	0.04-0.4 μl
PCR-grade Water	-	fill up to 20 μl

Incubate for 5 min for cohesive-ended ligations or 15 min for blunt-ended ligations at ambient temperature (20 - 25 °C).

BIOZ Product Citations:

T4 DNA Ligase

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