|Molecular Biology||Storage: -20°CShipping: Blue Ice|
5'-A T T T^A A A T-3'
3'-T A A A^T T T A-5'
Streptococcus milleri S
One unit is defined as the amount of SmiI required to digest 1ug of Ad2 DNA-SspI fragments in 1 hour at 30°C in 50ul of assay buffer.
10mM Tris-HCl (pH 7.4, 25°C) 200mM NaCl, 1mM DTT, 1mM EDTA, 1mM DTT, 0.2mg/ml BSA, 50% glycerol.
R1625 Restriction Enzyme Buffer A: 10X: 10mM Tris-HCl (pH 7.4, 25°C) 100mM KCl, 1mM EDTA, 1mM DTT, 0.2mg/ml BSA, 50% glycerol. For longer periods, the Storage Buffer should be used.
R1625-03 Restriction Enzyme Buffer D: 10X: 50mM Tris-HCl, pH 7.5, 10mM MgCl2, 100mM sodium chloride, 0.1mg/ml BSA.
Storage and Stability:
May be stored at 4°C for short-term only. Aliquot to avoid repeated freezing and thawing. Store at -20°C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.
No detectable change in the specific fragmentation pattern was observed after 160-fold overdigestion (10u/ug Ad2 DNA x 16 hours) with S1014-59.
After 50-fold overdigestion (3u/ug DNA x 17 hours) with S1014-59, more than 80% of the DNA fragments can be ligated in a reaction mixture containing 20–40u of T4 DNA Ligase/1ug of fragments and 10% PEG at a 5'-termini concentration of 0.06uM. More than 95% of these can be recut.
Labeled Oligonucleotide (LO) Assay:
No detectable degradation of a single-stranded and double-stranded labeled oligonucleotide occured during incubation with 10 units of S1014-59 for 4 hours.
Blue/White Cloning Assay:
The mix of pUC57/HindIII, pUC57/Eco32I and pUC57/PstI digests was incubated with 10 units of SmiI for 16 hours. After religation and transformation, the background level of white colonies was <1%.
30ºC. Incubation at 37ºC results in 70% activity.
Dam, Dcm, CpG, EcoKI: never overlaps; no effect.
EcoBI: may overlap; effect not determined
Stability during Prolonged Incubation:
A minimum of 0.1 unit of SmiI is required for complete digestion of 1ug of Ad2 DNA in 16 hours at 30°C
SmiI is inactivated by incubation at 65°C for 20min.
Digestion of Agarose-embedded DNA:
A minimum of 10 units of SmiI is required for digestion of 1ug of agarose-embedded Ad2 DNA in 16 hours.
Number of Recognition Sites in DNA: