T7 Endonuclease I – LE101

T7 Endonuclease I

T7 Endonuclease I is a junction-resolving enzyme of 149 amino acid residues. T7 endo I is encoded by gene 3 of bacteriophage T7 and exists as a stable dimer. Not only does it selectively bind and cleave four-way DNA (Holliday) junctions with high-specificity for branched structures in double-stranded DNA (such as cruciform DNA) but also it has a strong preference for cutting single-stranded DNA. It requires metal ions such as magnesium for full enzymatic activity.

Applications for T7 endonuclease I

• Gene mutation and SNP detection in TALEN and CRISPR/CAS9 engineered specimens;
• Recognition and cleavage of non-perfectly matched DNA and Holliday junctions;
• Random cleavage of single-stranded DNA.

Source

T7 endonuclease I is purified from E. coli expressing the recombinant T7 Endonuclease I (T7EI) gene.

Unit Definition

One unit is defined as the amount of enzyme required to convert > 90% of 1 μg of supercoiled cruciform pUC(AT) to > 90% linear form in a total reaction volume of 50 μl in 1 hour at 37°C.

Quality Control

Exonuclease activity: 10 units of enzyme are incubated with 1 μg of [³H] labeled DNA (purified PCR products) at 37°C for 2 hour in 100 μl reaction system, and < 5% radioactive material is released.
Endonuclease activity: 5 units of T7 endo I are incubated with 1 μg of pBR322 DNA at 37°C for 2 hours in 50 μl reaction system, and the ratio of RF I to RF II is no more than 20%.

Contents

Storage

at -20 °C for one year.

Manual and datasheet

LE101 – T7 endonuclease I