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Production Description

KAPA T4 DNA ligase catalyzes the formation of a phosphodiester bond between 5’ phosphate and 3’ hydroxyl termini in duplex DNA or RNA. This enzyme will join blunt and cohesive end termini as well as repair single stranded nicks in duplex DNA, RNA or DNA/RNA hybrids1.

Product Applications

KAPA T4 DNA Ligase is ideally suited for:

Ligation Protocol

In a microcentrifuge tube, combine the following reagents:

Vector DNA	x µ(10-100 ng)
Insert DNA	x µl*
10X ligation buffer	2µl
Sterile ddH2O	Up to 20 µl
KAPA T4 DNA Ligase	1µl (400U or 2,000 U)

Vortex the tube and briefly centrifuge.

Incubate the mixture for 1 hour at room temperature.

Immediately transform competent cells** with 2 µl of the ligation reaction.

*For cohesive-end ligations, use a 1:1 or a 3:1 molar ratio of insert:vector; for blunt-end ligations, use a 3:1 molar ratio of insert:vector DNA.

**Transformation can be done using chemically-competent or electrocompetent cells. Electrocompetent cells may show significantly higher transformation efficiency (several logs higher).

Basic Transformation Protocol

*** SOC media: 2% Bactotryptone, 0.5% Yeast extract, 2.5mM KCl, 10mM NaCl, 10mM MgSO4, 10mM MgCl2 and 20mM Glucose.

Quality Control

SDS-PAGE
2.0 µl (10.0 µg) of enzyme solution was loaded on a denaturing 4-20% Tris-Glycine SDS-PAGE gel flanked by a broad-range MW marker and 2.0 µl (100 ng) of a 1:100 dilution of the sample. Following electrophoresis, the gel was stained using the silver stain technique (Invitrogen SilverQuest). The aggregate mass of contaminant bands in the concentrated sample did not exceed the mass of the protein of interest band in the dilute sample, confirming greater than 99% purity of the concentrated sample.

Single-Stranded Exonuclease Assay
A 50 µl reaction containing 50,000 cpm of tritiated oligo dT and 10 µl (50,000 U) of enzyme solution incubated for 16 hours at 37ºC resulted in less than 0.1% release of TCA-soluble counts.

Double-Stranded Exonuclease Assay
A 50 µl reaction containing 15,000 cpm of a 1 kb, tritiated, double stranded DNA fragment and 10 µl (50,000 U) of enzyme solution incu- bated for 4 hours at 37ºC resulted in less than 0.1% release of TCA-soluble counts.

Endonuclease Activity
A 50 µl reaction containing 1 µg of pBR322 DNA and 10 µl (50,000 U) of enzyme solution incubated for 4 hours at 37ºC resulted in no visually discernible conversion to nicked circular DNA as determined by agarose gel electrophoresis.

Real-Time PCR DNA Contamination Test
Replicate 5 µl samples were heat denatured and screened in a TaqMan® qPCR assay for the presence of contaminating E.coli genomic DNA using primers for the 16S rRNA locus. The absolute quantification method of detection was employed, with serial dilutions of purified E.coli K-12 used to draw a standard curve (5 points, R2=0.991). Based on no template control Ct values, the detection limit of this assay is <10 copies genome/sample. Replicate average samples was observed to be <10 copies of genome/25,000 U T4 DNA Ligase.

Unit Characterization Assay
Unit activity was measured using a 2-fold serial dilution method. Dilutions of enzyme batch were made in 1X KAPA T4 DNA Ligase Reaction Buffer ([T4 DNA Ligase]f = 0.31-20 µg/µl) and added to 50 µl reactions containing 0.1 µg DNA and 1X KAPA T4 DNA Ligase Reaction Buffer. Reactions were incubated 30 minutes at 23ºC (room temp), plunged on ice, and analyzed on a 1% agarose gel stained with Ethidium Bromide. 1 unit is defined as the amount of KAPA T4 DNA Ligase required to ligate 50% of 100 ng DNA fragments with cohesive termini in
50 µl following a 30 minute incubation at 23ºC.

References

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

Limitations on Use

This product was developed, manufactured, and sold for research and in vitro use only. The product is not suitable for administration to humans or animals. MSDS sheets relevant to this product are available upon request.

 

	KAPA T4 Ligase (both concentrations) supplied in 10 mM Tris-HCl, 50 mM KCl, 1 mM Dithiothreitol, 0.1 mM EDTA, 50% Glycerol, pH 7.4 @ 25°C
	10x KAPA T4 Ligase Reaction Buffer