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 37C. The target DNA substrate can be cleaved and detected with 15 minutes at 37C. 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 37C. 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 37C. 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 4C for short-term only. Aliquot to avoid repeated freezing and thawing. Store at -20C. Aliquots are stable for 6 months after receipt at -20C. 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.
Reinheit:
95%
Formulierung:
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
* Mehrwertsteuer und Versandkosten nicht enthalten. Irrtümer und Preisänderungen vorbehalten
