|Molecular Biology||Storage: -20°CShipping: Blue Ice|
5'-^ 8(N) G A G (N)5 C T C (N)13^-3'
3'-^13(N) C T C (N)5 G A G (N) 8^-5'
BplI requires only Mg2+ for its activity, but is stimulated by S-adenosylmethionine. 0.05mM S-adenosylmethionine gives more than a 100-fold increase of BplI activity. The cleavage of DNA by BplI is never complete.
One unit is defined as the amount of Bpl I required to digest 1ug of lambda DNA-Xhol in 1 hour at 37°C in 50ul of assay buffer (a maximal cleavage level is achieved at which no change in the fragmentation pattern is observed with further increase of enzyme).
Supplied as a liquid in 10mM Tris-HCl, pH 7.5, 100mM KCl, 1mM DTT, 1mM EDTA, 0.2mg/ml BSA, 50% glycerol.
R1625 Restriction Enzyme Buffer A, 10X:
33mM Tris-acetate, pH 7, 10mM magnesium acetate, 66mM potassium acetate, 0.1mg/ml BSA
Storage and Stability:
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.
Stability during Prolonged Incubation:
A minimum of 0.3 units of Bpl I is required for complete digestion of 1ug of DNA in 16 hours at 37°C.
Bpl I is inactivated by incubation at 65°C for 20min.
DAM, DCM, EcoKl, EcoBl: never overlaps - no effect
CpG: may overlap - no effect
Number of Recognition Sites in DNA:
PhiX174, M13mp18/19,pBR322, pUC18/19, pUC57, pTZ19R/U: 0
Digestion of Agarose-embedded DNA:
A minimum of 10 units of the Bpl I is required for complete digestion of 1ug of agarose-embedded lambda DNA in 16 hours.
No detectable change in the specific fragmentation pattern is observed after 160-fold overdigestion (10u/ug lambda DNA x 16 hours) with BplI.
After 10-fold overdigestion (0.6u/ug DNA x 17 hours) with BplI, more than 70% of the DNA fragments can be ligated at a 5'-termini concentration of 0.03µM. None of these can be recut due to the methylation at the recognition sequence by Bpll.
Labeled Oligonucleotide (LO) Assay:
No detectable degradation of a single-stranded and double-stranded labeled oligonucleotide was observed after incubation with 10 units of Bpl I for 4 hours.
Blue/White Cloning Assay:
The mix of pUC57/HindIII, pUC57/PstI and pUC57/Eco32I digests was incubated with 10 units of Bpl I for 16 hours. After religation and transformation the background level of white colonies was <1%.