T4 DNA Ligase

What is T4 DNA Ligase?

T4 DNA Ligase (Product No. D2886) 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 The protein source is a recombinant E. coli strain carrying the cloned T4 DNA Ligase gene.

T4 DNA Ligase Applications

Common applications for T4 DNA include the following:

• Cloning of PCR products
• Cloning of restriction enzyme generated DNA fragments
• Joining of double-stranded oligonucleotide linkers or adaptors to DNA
• Site-directed mutagenesis
• Amplified fragment length polymorphism (AFLP)
• Ligase-mediated RNA detection
• Nick repair in duplex DNA, RNA or DNA/RNA hybrids
• Self-circularization of linear DNA

Usage Notes

One T4 DNA Ligase cohesive end unit is equivalent to approximately three 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. For best results, store the reaction buffer at -20 °C and discard after one year of storage.

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

Unit Definition of T4 DNA Ligase

One Weiss unit is defined as the amount of enzyme required to catalyze the exchange of 1 nmol of P³² from pyrophosphate into ATP as Norit-absorbable material in 20 minutes at 37 °C.

General T4 DNA Ligase Protocol

Reaction setup*:

* Total reaction volume can be adjusted as needed.

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

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

References

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