Descrição
AmpTherm DNA Ligase is a thermostable DNA ligase that catalyzes the formation of a phosphodiester bond between a 5´-phosphate and a 3´-hydroxyl group of two adjacent oligonucleotides hybridized to the same complementary target DNA strand. This ligation reaction occurs only when the two oligonucleotides are perfectly matched to the complementary target DNA and are directly adjacent to each other without any gaps. Therefore, it can be used for the detection of single-base substitutions. This enzyme requires ATP as a cofactor and exhibits activity over a temperature range of 45–70°C.
Product information
|
Catalog Number |
14959ES84 / 14959ES92 |
|
Size |
2,000 U / 10,000 U |
|
Gene Source |
Thermococcus sp. |
|
Expression Host |
E.coli |
|
Activity Definition |
One unit of activity (U) is defined as the amount of enzyme required to ligate 50% of 1 μg of BstEII-digested λDNA fragments (with 12 bp cohesive ends) in a 15-minute incubation at 45°C. |
|
Storage Buffer |
10 mM Tris-HCl, 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 200 µg/mL BSA, 50% Glycerol, 10 mM (NH4)2SO4, pH 7.4 @ 25°C |
|
Concentration |
40 U/µL |
Component information
|
Component No. |
Component Name |
14959ES84 |
14959ES92 |
|
14959-A |
AmpTherm DNA Ligase (40 U/µL) |
50 µL |
250 µL |
|
14959-B |
10× AmpTherm DNA Ligase Buffer |
200 µL |
1 mL |
Applications
1. Allele-specific detection via Ligase Detection Reaction (LDR) and Ligase Chain Reaction (LCR).
2. Ligation of phosphorylated oligonucleotides in Ligase Chain Reaction (LCR).
3. Sealing of nicks in DNA strands at elevated temperatures.
Storage conditions
This product should be stored at -25~-15℃ for 2 years.
Instructions for use
1. Reaction Setup:
|
Component |
Volume |
|
10× AmpTherm DNA Ligase Buffer |
2 µL |
|
Substrate: DNA with complementary cohesive ends >8 nt |
100 ng – 1 µg |
|
AmpTherm DNA Ligase (40 U/µL) |
1-2 µL |
|
ddH2O |
To 20 µL |
2. Reaction Conditions: Incubate at 45–50°C for 15 minutes.
3. Product Analysis: Analyze the products by agarose gel electrophoresis for longer double-stranded DNA substrates; use polyacrylamide gel electrophoresis for short DNA fragments (<100 bp).
Notes
1. This product can effectively ligate 12 bp complementary fragments, but cannot ligate 4 bp complementary fragments (typical products of restriction enzyme digestion).
2. This enzyme requires ATP as a cofactor for the reaction.
3. Keep the enzyme on ice or in an ice box during use. After use, store immediately at -25°C to -15°C.
4. For your safety and health, please wear a lab coat and disposable gloves when handling this product.
5. This product is for research use only!
Documents:
Safety Data Sheet
Manuals:
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