| 5'-C G C G-3' |
|
| 3'-G C G C-5' |
|
| Catalog # | B2922 |
| Source | Bacillus sphaericus RFL1236 |
| Concentration | 10u/ul |
| Incubation Temperature | 37°C |
| Storage Buffer | Supplied as a liquid in 10mM Tris-HCl, pH 7.5, 100mM KCl, 1mM DTT, 0.1mM EDTA, 0.2mg/ml BSA, 50% glycerol. |
| Diluent Buffer | 10mM Tris-HCl, pH 7.4, 100mM KCl, 1mM EDTA, 1mM DTT, 0.2mg/ml BSA and 50% glycerol. |
| Supplied with | R1625: Restriction Enzyme Buffer A, 10X: 33mM Tris-acetate, pH 7, 10mM magnesium acetate, 66mM potassium acetate, 0.1mg/ml BSA |
| R1625-04 | Restriction Enzyme Buffer E, 10X, 10mM Tris-HCl, pH 8.5 at 37°C, 10mM MgCl2, 100mM KCl, 0.1mg/ml BSA |
| Storage and Stability | Aliquot to avoid repeated freezing and thawing. Store at -20°C. Aliquots are stable for 6 months at -20°C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. |
| Enzyme Properties | |
| Methylation Effect | |
| Blocked by CpG methylation. |
| Dam, Dcm, EcoKl, EcoBl | Neber overlaps, no effect |
|
| Stability during Prolonged Incubation | |
| A minimum of 0.1units of B2922 is required for complete digestion of 1ug of lambda DNA in 16 hours at 37°C. |
|
| Thermal Inactivation | |
| B2922 is inactivated by incubation at 65°C for 20min. |
|
| Quality Control | |
| Overdigestion Assay | |
| No detectable change in the specific fragmentation pattern is observed after 160-fold overdigestion (10u/ug lambda DNA x 16 hours) with Bsh1236I. |
|
| Ligation/Recutting Assay | |
| After 50-fold overdigestion (3u/ug DNA x 17 hours) with Bsh1236I, more than 95% of the DNA fragments can be ligated at a 5'-termini concentration of 1.6uM. More than 95% of these can be recut. |
|
| Labeled Oligonucleotide (LO) Assay | |
| No detectable degradation of a single-stranded and double-stranded labeled oligonucleotide was observed after incubation with 10units of B2922 for 4 hours. |
|
| Number of Recognition Sites in DNA | |
| Lambda | 157 |
| PhiX174 | 14 |
| M13mp18/19 | 18 |
| pBR322 | 23 |
| pUC18/19 | 10 |
| pUC57 | 11 |
| pTZ19R/U | 11 |
|
| Protocol for Digestion | |
| Add | |
| Nuclease free water | 16ul |
| R1625-04 | 2ul |
| DNA (0.5-1ug/ml) | 1ul |
| B2922 | 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 |
| R1625-04 | 2ul |
| B2922 | 1-2ul |
| Mix gently and spin down for a few seconds. |
| Incubate at 37ºC for 1-16 hours. |
