|Molecular Biology||Storage: -20°CShipping: Blue Ice|
5'-G G^C C-3'
3'-C C^G G-5'
Bacillus subtilis R
Diluent Buffer :
10mM Tris-HCl, pH 7.4, 100mM KCl, 1mM EDTA, 1mM DTT, 0.2mg/ml BSA and 50% glycerol.
10mM Tris-HCl, pH 7.5, 50mM KCl, 1mM DTT, 0.1mM EDTA, 0.2mg/ml BSA and 50% glycerol.
R1625-Restriction Enzyme Buffer A, 10X: Dilute to 1X for use. 1X buffer composition is 33mM Tris-acetate (pH 7.9 at 37°C), 10mM magnesium acetate, 66mM potassium acetate, 0.1mg/ml BSA.
R1625-04: Dilute to 1X for 100% BsuRI digestion. 10mM Tris-HCl, pH 8.5, 10mM MgCl2, 100mM KCl and 0.1mg/ml BSA. Incubate at 37°C
Incubation Temperature: 37°C
One unit is defined as the amount of BsuRI required to digest 1ug of lambda DNA in 1 hour at 37°C in 50ul of assay buffer.
Protocol for Digestion:
Nuclease free water: 16ul
DNA (0.5-1ug/ml): 1ul
Mix gently and spin down for a few seconds. Incubate at 37ºC for 1-16 hours.
Protocol for Digestion Directly after Amplification:
PCR Reaction Mixture: 10ul (~0.1-0.5ug of DNA)
Nuclease free water: 18ul
Mix gently and spin down for a few seconds.
Incubate at 37ºC for 1-16 hours.
Storage and Stability:
Aliquot and 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.
No detectable change in the specific fragmentation pattern is observed after 160-fold overdigestion (10u/ug lambda DNA x 16 hours) with BsuRI.
After 50-fold overdigestion (3u/ug DNA x 17 hours) with BsuRI, more than 95% of the DNA fragments can be ligated at a 5'-termini concentration of 1.5uM. 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 BsuRI for 4 hours.
Stability during Prolonged Incubation:
A minimum of 0.1units off BsuRI is required for complete digestion of 1ug of lambda DNA in 16 hours at 37°C.
Number of Recognition Sites in DNA:
PhiX174, pUC18/19: 11