| 5-G T A C-3 3-C A T G-5 Unlike RsaI, Csp6I produces DNA fragments with a 2-base 5-extension. Source: Corynebacterium species RFL6 Concentration: 10u/ul Unit Definition: One unit is defined as the amount of enzyme required to digest 1ug of lambda DNA in 1 hour at 37C in 50ul of assay buffer. Storage Buffer: Supplied as a liquid in 10mM Tris-HCl, pH 7.4 at 25C, 100mM potassium chloride, 1mM DTT, 1mM EDTA, 0.2mg/ml BSA, 50% glycerol. Supplied With: R1625 Restriction Enzyme Buffer A, 10X: Supplied as a liquid in33mM Tris-acetate, 10mM magnesium acetate, 66mM potassium acetate, 0.1mg/ml BSA, pH 7.9 at 37C. R1625-01 Restriction Enzyme Buffer B, 10X: Supplied as a liquid in 10mM Tris-HCl, pH 7.5, 10mM magnesium chloride, 0.1mg/ml BSA. Incubate at 37C Enzyme Properties: Stability during Prolonged Incubation: A minimum of 0.1u of Csp6I is required for complete digestion of 1ug of lambda DNA in 16 hours at 37C. Thermal Inactivation: Csp6I is inactivated by incubation at 65C for 20min. Compatible Ends: FspBI, NdeI, Tru1I, VspI Number of Recognition Sites in DNA: Lambda: 113 PhiX174: 11 pBR322, pUC57, pUC18/19: 3 pTZ19R/U: 2 M13mp18/19: 19 Methylation Effects: Dam, Dcm, EcoKI, EcoBI: Never overlaps-no effect CpG: May overlap-no effect Quality Control: Overdigestion Assay: No detectable change in the specific fragmentation pattern is observed after 320-fold overdigestion (20u/ug lambda DNA x 16 hours) with Csp6I. Ligation/Recutting Assay: After 50-fold overdigestion (3u/ug DNA x 17 hours) with Csp6I more than 95% of the DNA fragments can be ligated at a 5-termini concentration of 05uM. More than 95% of these can be recut. Labeled Oligonucleotide Assay: No detectable degradation of a single-stranded and double-stranded labeled oligonucleotide was observed after incubation with 10u of Csp6I for 4 hours. Protocol for Digestion: Add: Nuclease free water: 16ul R1625-01: 2ul DNA (0.5-1ug/ml): 1ul C7946: 0.5-2ul Mix gently and spin down for a few seconds. Incubate at 37C for 1-16 hours. Protocol for Digestion Directly after Amplification: Add: PCR Reaction Mixture: 10ul (~0.1-0.5ug of DNA) Nuclease free water: 18ul R1625-01: 2ul C7946: 1-2ul Mix gently and spin down for a few seconds. Incubate at 37C for 1-16 hours. Storage and Stability: May be stored at 4C for short-term only. For long-term storage, 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. |
