Recombinant T4 DNA Ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5 -phosphate and 3 -hydroxyl termini in duplex DNA or RNA. This enzyme will join blunt end and cohesive end termini as well as repair single stranded nicks in duplex DNA, RNA or DNA/RNA hybrids.
Kit Components:
D3931-04A: T4 DNA Ligase 1x20Ku (120ul) Supplied as a liquid in 10mM Tris-HCl, pH 7.4, 50mM potassium chloride, 0.1mM EDTA, 1mM DTT, 0.2mg/ml BSA and 50% glycerol.
D3931-04B: T4 DNA Ligase Reaction Buffer (10X) 1x1ml. Supplied as a liquid as 500mM Tris-HCl, pH 7.5, 100mM magnesium chloride, 100mM DTT, 10mM ATP, 0.25mM BSA.
D3931-04C: 10mM ATP, 1x100ul
Reaction Conditions:
1X T4 DNA Ligase Reaction Buffer and DNA (0.1-1uM in 5� termini). Incubate at 16 degrees C.
Unit Definition:
1. One unit is defined as the amount of enzyme required to give 50% ligation of Hind III fragments of DNA (5� DNA termini concentration of 0.12uM, 300ug/ml) in a total reaction volume of 20ul in 30 minutes at 16 degrees C in 1X T4 DNA Ligase Reaction Buffer.
2. One Weiss unit is defined as the amount of enzyme required to catalyze the exchange of 1nmol of 32P from pyrophosphate to ATP, into Norit-adsorbable material in 20 minutes at 37 degrees C.
Activity:
One Weiss unit is equivalent to ~67 cohesive-end ligation units.
� T4 DNA Ligase is strongly inhibited by NaCl or KCl if the concentration is > 200mM.
� Ligation of blunt-ended and single-base pair overhang fragments requires about 50 times as much enzyme to achieve the same extent of ligation as cohesive-end DNA fragments. Blunt-end ligation may be enhanced by addition of PEG 4000 (10% w/v final concentration) or hexamine chloride, or by reducing the ATP concentration to 50uM.
Heat Inactivation:
T4 DNA Ligase can be inactivated by incubation at 65 degrees C for 10 minutes.
Quality Control:
Purified free of contaminating endonucleases and exonucleases. Each lot of T4 DNA ligase is also tested in a mock cloning assay, which reveals any damage to the ligated DNA termini. Greater than 99.9% of the termini remain undamaged in this assay.
Exonuclease Activity:
Incubation of a 50ul reaction containing 13,000 units of T4 DNA Ligase with 1ug of a mixture of single and double-stranded [3H] E. coli DNA (200,000cpm/ g) for 4 hours at 37C released <0.3% of the total radioactivity.
Endonuclease Activity:
Incubation of a 50ul reaction containing 13,000 units of T4 DNA Ligase with 1 g of X174 RF I DNA for 4 hours at 37C resulted in < 5% conversion to RFII as determined by agarose gel electrophoresis.
Nuclease Activity:
Incubation of 13,000 units for 18 hours in assay buffer (without ATP) with Hind III fragments of lambda DNA yielded a clear and sharp banding pattern on agarose gels.
Applications:
Suitable for use in cloning of restriction fragments, and joining linkers and adapters to blunt-ended DNA. Other applications not tested.
Storage and Stability:
May be stored at 4 degrees C for short-term only. Aliquot to avoid repeated freezing and thawing. Store at -20 degrees C. Aliquots are stable for at least 6 months at -20 degrees C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.
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