| 5'-G^T G C A C-3' |
|
| 3'-C A C G T^G-5' |
|
| Catalog # | A1372-15 |
| Unit Definition | One unit is defined as the amount of Alw44l required to digest 1ug of lambda DNA-Smal fragments in 1 hour at 37ºC in 50ul of assay buffer. |
| Diluent Buffer | 10mM Tris-HCl, pH 7.4, 100mM KCl, 1mM EDTA, 1mM DTT, 0.2mg/ml BSA, 50% glycerol. |
| Storage Buffer | Supplied as a liquid in 10mM Tris-HCl pH 7.5, 50mM KCl, 1mM DTT, 0.1mM EDTA, 0.2mg/ml BSA, 50% glycerol. |
| Supplied with | R1625-Restriction Enzyme Buffer A (double digests): 1X Buffer composition-Supplied as a liquid containing 33mM Tris-acetate pH 7.9, 10mM magnesium acetate, 66mM potassium acetate, 0.1mg/ml BSA. Incubate at 37°C. |
| Methylation Effects on Digestion | |
| Dam | Never overlaps - no effect. |
| Dcm | Never overlaps - no effect. |
| CpG | Completely overlaps - blocked |
| EcoKl | Never overlap - no effect |
| EcoBl | Never overlaps - no effect. |
|
| Number of Recognition Sites in DNA | |
| Lambda | 4 |
| PhiX174 | 1 |
| pBR322 | 3 |
| pUC57 | 3 |
| pUC18/19 | 3 |
| pTZ19R/U | 2 |
| M13/mp18/19 | 1 |
|
| Compatible Ends | |
| Bfml |
|
| Digestion of Agarose-embedded DNA | |
| A minimum of 5 units of the enzyme is required for complete digestion of 1ug of agarose-embedded lambda DNA in 16 hours. |
|
| Overdigestion Assay | |
| No detectable change in the specific fragmentation pattern is obseAlw44l.. |
| rved after 160-fold overdigestion (10u/ug lambda DNA x 16 hours) with Alw44I |
|
| Stability during Prolonged Incubation | |
| A minimum of 0.1 units of the Alw44I is required for complete digestion of 1ug of DNA in 16 hours at 37ºC. |
|
| Protocol for Digestion | |
| Add | |
| Nuclease free water | 16ul |
| R1625 | 2ul |
| DNA (0.5-1ug/ml) | 1ul |
| A1372-15 | 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 | 2ul |
| A1372-15 | 1-2ul |
| Mix gently and spin down for a few seconds. |
| Incubate at 37ºC for 1-16 hours. |
|
| Ligation/Recutting Assay | After 50-fold overdigestion (3u/ug DNA x 17 hours) with Alw44I more than 90% of the DNA fragments can be ligated at a 5'-termini concentration of 0.03uM. More than 95% of these can be recut. |
|
| Labeled Oligonucleotide (LO) Assay | No detectable degradation of a single-stranded and double-stranded labeled oligonucleotide occurred during incubation with 10 units of Alw44l for 4 hours. |
|
| Thermal Inactivation | Enzyme is inactivated by incubation at 65°C for 20 minutes. |
