other T4 DNA Bioactive Protein Product Attributes
Product Type: Bioactive Protein
Recombinant T4 DNA based upon sequence from other
Host: QP10872 protein expressed in E. Coli.
Available Tags: Untagged
Protein Construction: A cDNA sequence encoding the sequence of T4 DNA was constructred and used to recombinantly synthesize the protein.
Recommended Applications: Bioactive
Application Notes
Definition of Unit (for specific activity): 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 0.12 µM, 300- µg/ml) in a total reaction volume of 20 ul in 30 minutes at 16°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 1 nmol of 32P from pyrophosphate to ATP, into Norit-adsorbable material in 20 minutes at 37°C.
Other Applications: Incubation of a 50µl reaction containing 13,000 units of T4 DNA Ligase with 1µg of a mixture of single and double-stranded [3H] E. coli DNA (200,000 cpm/ug) for 4 hours at 37°C released < 0.3% of the total radioactivity. Incubation of 13,000 units for 18 hours in assay buffer (without ATP) with Hind III fragments of gamma DNA yielded a clear and sharp banding pattern on agarose gels. T4 DNA Ligase can be inactivated by incubation at 65°C for 10 minutes.
Additional Information: 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.
Bioactivity Data: Incubation of a 50µl reaction containing 13,000 units of T4 DNA Ligase with 1µg of X174 RF I DNA for 4 hours at 37°C resulted in < 5% conversion to RFII as determined by agarose gel electrophoresis. One Weiss unit is equivalent to circa 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 50µM. To dilute T4 DNA Ligase that will subsequently be stored at Â20°C, 50% glycerol storage buffer should be used; to dilute for immediate use, 1x T4 DNA Ligase reaction buffer can be used.
Reconstitution Instructions: |||
Endotoxin Levels: < 1.0 EU per ug protein as determined by the LAL method.
Buffer: 50mM KCl, 10mM Tris-HCl (pH 7.4), 0.1mM EDTA, 1mM DTT, 200 µg/ml BSA and 50% glycerol. Store at -20C.
Physical Appearance: Sterile filtered liquid formulation having a concentration of 167,000 U/ml.
Limitations and Performance Guarantee
enQuire Bio's products are guaranteed and approved for RESEARCH USE ONLY or manufacturing purposes. This product may not be used as a pharmaceutical agent, or other product intended for human consumption without meeting additional regulatory requirements. This product is guaranteed to work for a period of two years when stored at -70C or colder, and one year when aliquoted and stored at -20C.
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