T5 Exonuclease, 1000 U (for Nuclease/Topoisomerase)

Product Information

Product Introduction

T5 Exonuclease is derived from the T5 phage D15 gene and expressed by Escherichia coli. It is a nuclease that degrades double-stranded or single-stranded DNA in the 5'3' direction. T5 Exonuclease can start digestion from the end, gap or nick of linear or circular double-stranded DNA but cannot degrade supercoiled double-stranded DNA. It is mainly used for Gibson assembly or other seamless cloning, degradation of linear and nicked plasmid DNA, etc.

Source: Derived from T5 bacteriophage and expressed by recombinant E. coli.

Enzyme activity definition: 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 deoxyribonucleotide within 30 min is defined as one inactivation unit.

Purity and concentration: Purity detected by SDS-PAGE 95%; 10 U/μL.

Inactivation or Inhibition:  Final EDTA concentrations greater than 11 mM can inactivate the enzyme.

Enzyme storage buffer: 50 mM Tris-HCl, 100 mM NaCl, 1 mM DTT, 0.1 EDTA, 50% Glycerol, 0.1% Triton X-100, pH 7.5.

10х T5 Reaction buffer: 500mM Potassium Acetate, 200mM Tris-acetate, 100mM Magnesium Acetate, 1mM DTT, pH 7.9.

Figure 1. 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 300 bp DNA fragment and the amount of T5 Exonuclease indicated in the figure. After incubation at 37°C for 10 min, immediately put on ice and add EDTA at a final concentration of 11 mM to terminate the reaction. Take 10 μL of the reaction product for gel electrophoresis detection.

Storage and transportation

Transported with wet ice; stored at -20, valid for 12 months.

composition

Procedure

1. Configure the reaction system according to the following table:

2. After preparing the reaction system, mix thoroughly and incubate at 37°C for 10-30 min;

3. Immediately place the reaction in an ice bath and add EDTA to a final concentration of 11 mM to terminate the reaction.

Precautions

1. T5 Exonuclease has a certain selectivity for DNA substrates and shows different reactivity to different DNA substrates. Therefore, it is necessary to properly control the enzyme amount and reaction time.

2. T5 Exonuclease also has certain activity at 50, so it can be used for Ginson assembly.

3. The enzyme should be placed in an ice box and stored at -20 immediately after use.

4. For your safety and health, please wear a lab coat and disposable gloves when operating.

The product is for scientific research purposes only and not for clinical diagnosis!

https://www.servicebio.com/goodsdetail?id=12050