T7 Endonuclease I (T7E1)
CRISPR/Cas9
T7 Endonuclease I (T7E1) recognizes and cleaves non-perfectly matched DNA, cruciform DNA structures, Holliday structures or junctions, hetero duplex DNA and more slowly, nicked double-stranded DNA. The cleavage site is at the first, second or third phosphodiester bond that is 5´ to the mismatch. The protein is the product of T7 gene 3.
T7 Endonuclease I is a fusion protein produced from E. coli.
Applications
•Resolve four-way junction or branched DNA. •Detect or cleave hetero duplex and nicked DNA. •Randomly cleave linear DNA for shot-gun cloning. •Detect gene mutagenesis and SNPs, for cleavage efficiency assays induced by TALEN, CRISPR/CAS9 or other gene editing tools.
Bioactivity
>90% of 1ug of supercoiled crusiform pUC (AT) to >90% linear form in a total reaction volume of 50ul in 1 hour at 37ºC. The target DNA substrate can be cleaved and detected with 15 minutes at 37ºC.
Unit Activity
One unit is defined as the amount of enzyme required to convert >90% of 1ug of supercoiled crusiform pUC (AT) to >90% linear form in a total reaction volume of 50ul in 1 hour at 37ºC.
Note: pUC (AT) is derived from puc19 with a modification of the polylinker between the EcoR1 site and Pstl site.
EcoR1 Pstl GAATTCATATATATATATATATATATATATATATATATATATATACTGCAG
Activity Test
To test the activity of 506220, a control gRNA targeting HPRT, which is co-transfected with Cas9 protein into 293T cells. After 48 hours, cells were lysed for genome PCR to amplify the specific taget site. The PCR product (~200ng) was then annealed and incubated with 1ul of 506220 for 15 minutes at 37ºC. Loading buffer was then added to the reaction mixture directly and the cleavage efficiency was detected by agarose gel electrophoresis.
Supplied With
506220A: Reaction Buffer, 10X Supplied as a liquid in 100mM Tris-HCl, 500mM sodium chloride, pH 7.9, 100mM MgCl2, 10mM DTT
Storage and Stability
May be stored at 4°C for short-term only. Aliquot to avoid repeated freezing and thawing. Store at -20°C. Aliquots are stable for 6 months after receipt at -20°C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.
Form
Supplied as a liquid in 20mM Tris-HCl, pH 7.5, 200mM sodium chloride, 0.1mM EDTA, 1mM dithiothreitol, 0.15% Triton X-100, 50% glycerol
Important Note
This product as supplied is intended for research use only, not for use in human, therapeutic or diagnostic applications without the expressed written authorization of United States Biological.
