Product Number

• LG01 T4 DNA Ligase
• LG02 Fast T4 DNA Ligase

Storage conditions

Store at -20°C

Description

FAST T4 DNA Ligase is designed for the efficient ligation of cohesive-ended DNA inserts into plasmid vectors in just 5-10 minutes (blunt-ended inserts in as little as 15-30 minutes). FAST T4 DNA Ligase catalyzes the joining of two strands of DNA between the 5'-phosphate and the 3'-hydroxyl groups of adjacent nucleotides in either a cohesive-ended or blunt-ended configuration. The enzyme has also been shown to catalyze the joining of RNA to either a DNA or RNA strand in a duplex molecule but will not join single-stranded nucleic acids.

Biological Source

E. coli strain expressing a recombinant clone.

Molecular Weight

68kDa.

Requirements

Mg²⁺, ATP and DTT. The optimum concentration of Mg²⁺ is 10mM. Mn²⁺ may be substituted for Mg²⁺ but is only 25% as effective as Mg²⁺.

Inhibition

50% inhibition by greater than 150mM NaCl (activity measured at nicks. Other inhibitors include 0.2M K⁺, Cs⁺, Li⁺, NH₄⁺ and 1mM spermine.

Inactivation

Heat to 70°C for 10 minutes.

Components

FAST T4 DNA Ligase (200 U/μl)
2 X Ligation Buffer

Applications

Cloning of restriction fragments.
Joining linkers and adapters to blunt-ended DNA

Unit Definition

One unit of FAST T4 DNA Ligase 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.

Physical Purity

The purity is ≥90% as judged by SDS-polyacrylamide gel electrophoresis with Coomassie® blue staining.

Standard Applications

We recommend using a 1:1, 1:3 or 3:1 molar ratio of vector: insert DNA when cloning a fragment into a plasmid vector. These ratios will vary with other types of vectors, for example, cDNA and genomic cloning vectors. The following example illustrates the conversion of molar ratios to mass ratios for a 3.0kb plasmid and a 0.5kb insert DNA fragment.

Protocol

The following ligation reaction of a 3kb vector and a 0.5kb insert DNA uses a 1:1 vector: insert ratio. Typical ligation reactions use 100-200ng of vector DNA.

1. Assemble the following reaction in a sterile microcentrifuge tube:

vector DNA	100ng
insert DNA	17ng
2 X Ligation Buffer	5μl
T4 DNA Ligase	0.5–1μl

Nuclease-Free Water to final volume of 10μl

2. Incubate the reaction at room temperature for 5-10 minutes for cohesive-ended ligations, or 15-30 minutes for blunt-ended ligations.

Notes

1. Ligation reactions performed using the 2 X Ligation Buffer do not need to be cleaned up before transformation.
2. Concatamers may form as ligation products. The extent of concatamer formation depends on the vector:insert ratio, incubation temperature and incubation time. This should be taken into account when screening transformants.

Order

LG01	T4 DNA Ligase	200U/μl, with ligase buffer
LG02	Fast T4 DNA Ligase	200U/μl, with Fast ligase buffer, only 5 minute ligation