T5 Exonuclease
T5 Exonuclease
Cat. No.: G3463-1000U
Size: 1000U
Description
T5 Exonuclease is T5 exonuclease, derived from T5 bacteriophage D15 gene recombinantly expressed in Escherichia coli, and is an exonuclease that degrades double-stranded or single-stranded DNA in the 5'→3' direction. T5 Exonuclease can initiate digestion from the end, gap or nick of linear or circular double-stranded DNA but cannot degrade supercoiled double-stranded DNA. Mainly used for Gibson assembly or other seamless cloning, degradation of linear and nicked plasmid DNA, etc.
- Source : Derived from T5 phage, recombinantly expressed by Escherichia coli.
- Definition of enzyme activity : under the conditions of 37°C and 50 μL reaction volume, the amount of enzyme required to hydrolyze double-stranded DNA to produce 1 nmol of acid-soluble deoxyribonucleotides within 30 minutes is defined as an inactivation unit.
- Purity and concentration : SDS-PAGE detection purity ≥ 95%; 10 U/μL
- Inactivation or inhibition : Enzymes can be inactivated at final concentrations of EDTA greater than 11 mM.
- Enzyme storage buffer : 50 mM Tris-HCl, 100 mM NaCl, 1mM DTT, 0.1 EDTA, 50% Glycerol, 0.1% Triton X-100, pH7.5.
- 10X T5 Reaction Buffer : 500 mM Potassium Acetate, 200 mM Tris-acetate, 100 mM Magnesium Acetate, 1 mM DTT, pH 7.9.
Figure 1. Effect diagram of T5 Exonuclease digestion of linear dsDNA. In a 50 μL reaction system (50 mM Potassium acetate, 20 mM Tris-acetate, 10 mM Magnesium acetate, 1 mM DTT, pH 7.9), add 1 μg of a 300 bp DNA fragment and the amount of T5 Exonuclease marked in the figure, 37°C After incubation for 10 min, the reaction was immediately terminated by adding 11 mM EDTA to the final concentration. 10 μL of the reaction product was taken for gel electrophoresis detection.
Storage and Handling Conditions
Composition
| Component Number | Component | G3463 |
| G3463-1 | T5 Exonuclease | 100 μL |
| G3463-2 | 10х T5 Reaction buffer | 1mL |
| Product Manual | 1 copy | |
Steps
1. Configure the reaction system according to the following table:
| Component | Volume |
| T5 Exonuclease | 1 μL |
| 10х T5 Reaction buffer | 5 μL |
| DNA samples | ≤1μg |
| Nuclease Free Water | To 50 μL |
3. Immediately after the completion of the reaction, ice-bath and add EDTA to a final concentration of 11 mM to terminate the reaction.
Precautions
- T5 Exonuclease has certain selectivity to DNA substrates, and it shows different reactivity to different DNA substrates. Therefore, it is necessary to properly control the amount of enzyme and the reaction time.
- T5 Exonuclease also has certain activity at 50°C, so it can be used for Ginson assembly.
- All enzymes should be stored in an ice box and stored at -20°C immediately after use.
- For your safety and health, please wear a lab coat and disposable gloves for operation.
