| 5'-C^C G G-3' |
|
| 3'-G G C^C-5' |
|
| Catalog # | H7030 |
| Concentration | 10u/ul |
| Source: Haemophilus parainfluenzae |
| Restriction Enzyme Buffer A, 10X (for 100% digestion) | 33mM Tris-acetate (pH 7.9), 10mM magnesium acetate, 66mM potassium acetate and 0.1mg/ml BSA. Incubate at 37°C. |
| Diluent Buffer | 10mM Tris-HCl (pH 7.4 at 25°C), 100mM KCl, 1mM EDTA, 1mM DTT, 0.2mg/ml BSA and 50% glycerol, or Storage Buffer. |
| Form | Supplied as a liquid in 10mM Tris-HCl (pH 7.4 at 25°C), 100mM NaCl, 1mM DTT, 0.1mM EDTA, 0.2mg/ml BSA and 50% glycerol. |
| 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 6 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. |
| Overdigestion Assay | No detectable change in the specific fragmentation pattern is observed after 160-fold overdigestion with HpaII(10u/ug lambda DNA x 16 hours) . |
|
| Ligation/Recutting Assay | After 50-fold overdigestion (3u/ug DNA x 17 hours) with HpaII, more than 95% of the DNA fragments can be ligated at a 5'-termini concentration of 3.1uM. 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 10 units of restriction endonuclease for 4 hours. |
|
| Methylation Effects | Dam, Dcm, EcoKI, EcoBI: never overlaps, no effect. |
| CpG | Completely overlaps, blocked |
|
| Stability during Prolonged Incubation | A minimum of 0.1 units of enzyme is required for complete digestion of 1ug of lambda DNA in 16 hours at 37°C. |
|
| Protocol for Digestion | |
| Add | 44ul nudlease free dH2O |
| 5ul Restriction Enzyme Buffer A, 10X |
| 1ul DNA (0.5-1ug/ul) |
| 0.5-2ul Hpall |
| Mix gently and spin down for a few seconds. |
| Incubate at 3ºC for 1-2 hours. |
|
| Protocol for Digestion of PCR products Directly after Amplification | |
| Add | 10ul (~1ug DNA) |
| 16ul nuclease free dH2O |
| 2ul Restriction Enzyme Buffer A, 10X |
| 1-2ul Hpall |
| Mix gently and spin down for a few seconds. |
| Incubate at 3ºC for 1-16 hours. |
|
| Thermal Inactivation | Enzyme is inactivated by incubation at 65°C for 20min. |
|
| Compatible Ends | AciI, Bsp119I, Bsu15I, Hin1I, MaeII, NarI, Psp1406I, TaqI, XmiI |
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| Number of Recognition Sites in DNA | |
|
| Lambda | 328 |
| PhiX174 | 5 |
| pBR322 | 26 |
| pUC57 | 13 |
| pUC18/19 | 13 |
| pTZ19R/U | 12 |
| M13mp18/19 | 18 |
|
| 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. |
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