T4 DNA Ligase

Catalog Number KEM0020
Storage Temperature –20 °C
Unit Size 150,000 U

Product Description

T4 DNA Ligase catalyzes the formation of a phosphodiester bond between the terminal 5′ phosphate and a 3′ hydroxyl groups of duplex DNA or RNA. The enzyme efficiently joins blunt and cohesive ends and repairs single stranded nicks in duplex DNA, RNA or DNA/RNA hybrids (1).

Source of Protein

A recombinant E. coli strain carrying the cloned T4 DNA Ligase gene.

Reagent

Supplied at a concentration 120,000 U/mL in 10 mM Tris-HCl, 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 50% glycerol, pH 7.5

Supplied with:

10X T4 DNA Ligase Buffer
Catalog Number KEM0049B
500 mM Tris-HCl, 100 mM MgCl2, 50 mM DTT,
10 mM ATP, pH 7.6

Precautions and Disclaimer

This product is for R&D use only, not for drug, household, or other uses. Please consult the Safety Data Sheet for information regarding hazards and safe handling practices.

Unit Definition

1 unit is defined as the amount of DNA Ligase required to join 50% of 100 ng of DNA fragments with cohesive termini in 50 µL 1X DNA Ligase Buffer following a 30 minute incubation at 23°C.

Protocol

Reaction setup*:

Component Volume (µL) Final Concentration
10X T4 DNA Ligase Buffer 2 µL 1X
Vector 10 1x X µL 1-10 ng/µL
Insert X µL 1-10 ng/µL
T4 DNA Ligase (120 U/µL) 1 µL 6 U/µL
Sterile Water X µL N/A
Total Volume 20 µL  

* Total reaction volume can be adjusted as needed.

1. Add all of above components to a clean reaction vessel, mix well by pipetting.
2. Incubate at 25 °C for 30 minutes.
3. Immediately purify DNA using PCR clean-up column and elute in ~50 µL.
4. OR - Immediately dilute (at least 1:10, but enough such that 0.1-10 ng of ligation product will be transformed) in TE or water
5. Transform 0.1-10 ng of ligation product into chemically or electrocompetent cell line that is compatible with vector

Usage Notes

One T4 DNA Ligase cohesive end unit is equivalent to approximately 3 cohesive end units as measured with a Lambda-Hind III DNA fragment substrate in 1X T4 DNA Ligase reaction buffer.

One Weiss Unit is approximately equivalent to 22 units.

T4 DNA Ligase is ATP dependent. It is recommended that the reaction buffer be discarded after one year of storage at –20 °C and replaced with fresh buffer to ensure maximum performance.

Single-insert ligations are optimal when targeting an insert:vector ratio between 2 and 6. A ratio above 6:1 will promote the insertion of multiple fragments, while dropping below 2:1 will reduce ligation efficiency.

Materials

     

Reference

1. Engler, M.J. and Richardson, C.C. (1982) P.D. Boyer (Eds.), The Enzymes, 5, pp. 3. San Diego: Academic Press.

 

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