| 5'-G^T A C-3' |
|
| 3'-C A T^G-5' |
|
| Unlike RsaI, Csp6I produces DNA fragments with a 2-base 5'-extension. |
|
| Catalog # | C7946 |
| 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 37°C in 50ul of assay buffer. |
| Storage Buffer | Supplied as a liquid in 10mM Tris-HCl, pH 7.4 at 25°C, 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 37°C. |
| 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 37°C |
| 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 37°C. |
| Thermal Inactivation: Csp6I is inactivated by incubation at 65°C 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 37ºC 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 37ºC for 1-16 hours. |
|
| Storage and Stability | |
| May be stored at 4°C for short-term only. For long-term storage, store at -20°C. Aliquots are stable for at least 12 months 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. |
