| 5' - G W G C W^C - 3' |
|
| 3' - C^W C G W G - 5’ |
|
| Catalog # | A1372-05 |
| Source | Acinetobacter lwoffi RFL21 |
| Concentration | 10u/ul |
| Form | Supplied as a liquid in 10mM Tris-HCl pH 7.5, 300mM sodium chloride, 1mM DTT, 0.1mM EDTA, 0.2mg/ml BSA, 50% glycerol. |
| Dilution Buffer | 10mM Tris-HCl pH 7.4, 100mM potassium chloride, 1mM DTT, 1mM EDTA, 0.2mg/ml BSA, 50% glycerol. |
| Unit Definition | One unit is defined as the amount of Alw21I required to digest 1ug of lambda DNA in 1 hour at 37°C in 50ul of assay buffer. |
| Supplied With | R1625: Restriction Enzyme Buffer A, 10X: Supplied as a liquid in 33mM Tris-acetate, 10mM magnesium acetate, 66mM potassium acetate, 0.1mg/ml BSA pH 7.9 at 37°C when diluted to 1X. Note: 1X conc. yields 20-50% enyzme activity. 2X conc. yields 50-100% enzyme activity. |
| R1625-03 | Restriction Enzyme Buffer D, 10X: Supplied as a liquid in 50mM Tris-HCl, pH 7.5, 10mM MgCl2, 100mM sodium chloride, 0.1mg/ml BSA when diluted to 1X. Incubate at 37°C. Note: This buffer yields 100% enzyme activity |
| Storage and Stability | May be stored at 4°C for short-term only. 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. |
| Overdigestion Assay | |
| No detectable change in the specific fragmentation pattern is observed after 160-fold overdigestion (10u/ug lambda DNA x 16 hours) with Alw21I. |
|
| Ligation/Recutting Assay | |
| After 50-fold overdigestion (3u/ug DNA x 17 hours) with Alw21I, more than 95% of the DNA fragments can be ligated at a 5'-termini concentration of 0.27uM. More than 95% of these sites 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 Alw21I for 4 hours. |
|
| Stability During Prolonged Incubation | |
| A minimum of 0.1 units of Alw21I is required for complete digestion of 1ug of lambda DNA in 16 hours at 37°C. |
|
| Compatible Ends | |
| GTGCA^C - BseSI, SduI, Mph1103I, PstI, SdaI |
| GAGCT^C - Eco24I, SacI, SduI |
| GAGCA^C - SduI |
|
| Number of Recognition Sites in DNA | |
| Lambda | 28 |
| PhiX174 | 3 |
| M13mp18/19 | 3 |
| pBR322 | 8 |
| pUC18/19 | 5 |
| pUC57 | 5 |
| pTZ19R/U | 4 |
|
| Thermal Inactivation | |
| Alw21I is inactivated by incubation at 65°C for 20 min. |
|
| Recommended Protocol for Digestion | |
| Add | |
| Nuclease free water | 16ul |
| 10X Restriction Enzyme Buffer D | 2ul |
| DNA (0.5-1ug/ul) | 1ul |
| Alw21I | 0.5-2ul |
| Mix gently, spin down for a few seconds. |
| Incubate at 37ºC for 1-16 hours. This digestion reaction may be scaled either up or down. |
|
| Protocol for Digestion of PCR Products Directly After Amplification | |
| Add | |
| PCR Reaction Mixture | 10ul (~0.1-0.5ug of DNA) |
| Nuclease free water | 18ul |
| 10X Restriction Enzyme Buffer D | 2ul |
| Alw21I | 1-2ul |
| Mix gently, spin down for a few seconds. |
| Incubate at 37ºC for 1-16 hours. |
|
| Methylation Effects on Digestion | |
| Dam, Dcm | Never overlaps, no effect |
| CpG, EcoKI | May overlap, no effect |
| EcoBI | May overlap, effect not determined |
