T4 DNA Ligase Active Protein | T4 DNA active protein
Recombinant T4 DNA Ligase
Synonyms
T4 DNA Ligase; Recombinant T4 DNA Ligase; T4 DNA; T4 DNA Ligase Recombinant; DNA ligase 4; EC 6.5.1.1; DNA ligase IV; Polydeoxyribonucleotide synthase [ATP] 4; T4 DNA active protein
Host
Escherichia Colilambda lysogen NM 989
Form/Format
(167,000 U/ml) 50mM Tris-HCl (pH 7.8 at 25°C), 10mM MgCl2, 10mM DTT, 1mM ATP, 25 ug/ml BSA and DNA (0.1 to 1 um in 5´ termini).
Optimal ligation occurs at 16°C.
Optimal ligation occurs at 16°C.
Application Notes
Cloning of restriction fragments.
Joining linkers and adapters to blunt-ended DNA.
Joining linkers and adapters to blunt-ended DNA.
Physical Appearance
Sterile filtered liquid formulation
Storage Buffer
50mM KCI, 10mM Tris-HCI (pH 7.4),0.1 mM EDTA, 1mM DTT, 200 ug/ml BSA and 50% glycerol. Store at -20°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 0.12 uM, 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.
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.
Biological Activity
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 50uM.
• To dilute T4 DNA Ligase that will subsequently be stored at –20°C, 50% glycerol storage buffer should be used; to dilute for immediate use, 1x T4 DNA Ligase reaction buffer can be used.
• 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.
• To dilute T4 DNA Ligase that will subsequently be stored at –20°C, 50% glycerol storage buffer should be used; to dilute for immediate use, 1x T4 DNA Ligase reaction buffer can be used.
Heat Inactivation
T4 DNA Ligase can be inactivated by incubation at 65°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 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.
Endonuclease Activity:
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.
Nuclease Activity :
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.
Exonuclease Activity:
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.
Endonuclease Activity:
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.
Nuclease Activity :
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.
Preparation and Storage
Two years when stored at -20°C, 2 weeks at 4°C.
Related Product Information for T4 DNA active protein
Description: 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.
Similar Products
Product Notes
The T4 DNA (Catalog #AAA142427) is an Active Protein produced from Escherichia Colilambda lysogen NM 989 and is intended for research purposes only. The product is available for immediate purchase. Cloning of restriction fragments. Joining linkers and adapters to blunt-ended DNA. Researchers should empirically determine the suitability of the T4 DNA for an application not listed in the data sheet. Researchers commonly develop new applications and it is an integral, important part of the investigative research process. It is sometimes possible for the material contained within the vial of "T4 DNA Ligase, Active Protein" to become dispersed throughout the inside of the vial, particularly around the seal of said vial, during shipment and storage. We always suggest centrifuging these vials to consolidate all of the liquid away from the lid and to the bottom of the vial prior to opening. Please be advised that certain products may require dry ice for shipping and that, if this is the case, an additional dry ice fee may also be required.Precautions
All products in the AAA Biotech catalog are strictly for research-use only, and are absolutely not suitable for use in any sort of medical, therapeutic, prophylactic, in-vivo, or diagnostic capacity. By purchasing a product from AAA Biotech, you are explicitly certifying that said products will be properly tested and used in line with industry standard. AAA Biotech and its authorized distribution partners reserve the right to refuse to fulfill any order if we have any indication that a purchaser may be intending to use a product outside of our accepted criteria.Disclaimer
Though we do strive to guarantee the information represented in this datasheet, AAA Biotech cannot be held responsible for any oversights or imprecisions. AAA Biotech reserves the right to adjust any aspect of this datasheet at any time and without notice. It is the responsibility of the customer to inform AAA Biotech of any product performance issues observed or experienced within 30 days of receipt of said product. To see additional details on this or any of our other policies, please see our Terms & Conditions page.Item has been added to Shopping Cart
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