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T4 DNA Ligase

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.

Supplied In
10 mM Tris-HCl
50 mM KCl
1 mM DTT
0.1 mM EDTA
50% glycerol
pH 7.5 @ 25°C

Supplied With
B6030 (10X T4 DNA Ligase Buffer)

10X T4 DNA Ligase Buffer (B6030)
500mM Tris-HCI
100 mM MgCl2
50 mM DTT
10 mM ATP
pH 7.6 @ 25°C

Unit Definition
1 unit is defined as the amount of T4 DNA Ligase required to join 50% of 100 ng of DNA fragments with cohesive termini in 50 µl 1X T4 DNA Ligase Buffer following a 30 minute incubation at 23°C.

Quality Control Analysis

Unit Characterization Assay
Unit activity was measured using a 2-fold serial dilution method. Dilutions of enzyme were made in 1X T4 DNA Ligase Reaction Buffer and added to 20 µL reactions containing double stranded DNA fragments and 1X T4 DNA Ligase Reaction Buffer. Reactions are incubated for 30 minutes at 23°C, stopped, and analyzed on a 1% agarose gel stained with ethidium bromide.

Protein Concentration (OD280) Measurement
A 2.0 µL sample of enzyme was analyzed at OD280 using a Nanodrop ND-1000 spectrophotometer standardized using a 2.0 mg/ml BSA sample (Pierce Cat #23209) and blanked with product storage solution. The observed average measurement of 3 replicate samples was converted to mg/mL using an extinction coefficient of 54,050 and molecular weight of 55,292 Daltons.

SDS-Page (Physical Purity Assessment) and Specifications
2.0 µL of concentrated 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 of a 1:100 dilution of the same enzyme species. Following electrophoresis, the gel was stained and the samples compared to determine physical purity. The acceptance criteria for this test requires that the aggregate mass of contaminant bands in the concentrated sample do not exceed the mass of the protein of interest band in the dilute sample, confirming greater than 99% purity of the concentrated sample.

Contamination Tests

Single-Stranded Exonuclease Activity
A 50 µL reaction containing 10,000 cpm of a radiolabeled single-stranded DNA substrate and 10 µL of enzyme solution incubated for 4 hours at 37°C resulted in less than 1.0% release of TCA-soluble counts.

Double-Stranded Exonuclease Activity
A 50 µl reaction containing 5,000 cpm of a radiolabeled double-stranded DNA substrate and 10 µL of enzyme solution incubated for 4 hours at 37&C resulted in less than 1.0% release of TCA-soluble counts.

Endonuclease Activity
A 50 µL reaction containing 0.5 µg of pENZuC DNA and 10 µL of enzyme solution incubated for 4 hours at 37°C resulted in no visually discernible conversion to nicked circular DNA as determined by agarose gel electrophoresis.

E.coli 16S rDNA Contamination Test:
Replicate 5 µL samples of enzyme solution were denatured and screened in a TaqMan qPCR assay for the presence of contaminating E.coli genomic DNA using oligonucleotide primers corresponding to the 16S rRNA locus. The acceptance criterion for the test is the threshold cycle count (Ct) produced by the average of 3 replicate no template control samples. Based on the correlation between the no template control Ct values and standard curve data, the detection limit of this assay is <10 copies genome/sample.

Product Information

T4 DNA Ligase
Part NumberL6030-LC-L
Price$363
Concentration120,000 U/ml
Unit Size150,000 U
SDS Available on request

Product Specification*

Storage Temperature-25 to -15°C
Test Specification
Purity (SDS-PAGE)>99%
Specific Activity300,000 U/mg
SS Exonuclease6,000 U <1.0% released
DS Exonuclease6,000 U <1.0% released
DS Endonuclease6,000 U = no conversion
E.coli DNA Contamination6,000 U <10 copies

* For a detailed summary of assay conditions and data, refer to the Quality Controls Analysis section.

Usage Instructions

Reaction Set-Up:

Amount Description Final Concentration
2 µL 10X T4 DNA Ligation Buffer 1X
X µL Vector 1-10 ng/µL
X µL Insert 1-10 ng/µL
1 µL T4 DNA Ligase (120 U/ µL) 6 U/µL
X µL Type I Water N/A
20 µL Total Volume

 

  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

Notes

One Enzymatics 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 Enzymatics cohesive end 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. For problematic ligations or if the DNA concentration is unknown, it may be necessary to vary ratios and run multiple ligations. Enzymatics 10X T4 DNA Ligase Buffer does not contain PEG and is compatible with standard ligation protocols which do not specify the use of a rapid/fast/quick format buffer.

References

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

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

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