KpnI
5'-G G T A C^C-3' 3'-C^C A T G G-5'
Source
Klebsiella pneumoniae OK8
Unit Definition
One unit is defined as the amount of KpnI required to digest 1ug of lambda DNA-BamHI fragmants 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. R1625-65: Restriction Enzyme Buffer for KpnI, 10X: Supplied as a liquid in 10mM Tris-HCl, pH 7.5, 10mM MgCl2, 0.02% Triton X-100 and 0.1mg/ml BSA. Incubate at 37°C.
Dilution Buffer
10mM Tris-HCl, pH 7.4, 100mM KCl, 1mM EDTA, 1mM DTT, 0.2mg/ml BSA and 50% glycerol.
Storage Buffer: Supplied as a liquid in10mM Tris-HCl (pH 7.5 at 25°C), 50mM KCl, 1mM DTT, 0.1mM EDTA, 0.2mg/ml BSA and 50% glycerol.
Thermal Inactivation
KpnI is inactivated by incubation at 80°C for 20min.
Overdigestion Assay: No detectable change in the specific fragmentation pattern is observed after 80-fold overdigestion (10u/ug lambda DNA x 16 hours) with KpnI.
Ligation/Recleavage (L/R) Assay: The ligation and recleavage assay was replaced with LO test after validating experiments showed LO test ability to trace nuclease and phosphatase activities with sensitivity that is higher than L/R by a factor of 100.
Labeled Oligonucleotide (LO) Assay
No detectable degradation of single- stranded or double-stranded labeled oligonucleotide was observed after incubation with 10 units of KpnI for 4 hours.
Blue/White Cloning Assay
The B/W assay was replaced with LO test after validating experiments showed LO test ability to detect nuclease and phosphatase activities with sensitivity that equals to that of B/W test.
Stability during Prolonged Incubation: A minimum of 0.2 units of KpnI is required for complete digestion of 1ug of lambda DNA in 16 hours at 37°C.
Lambda: 2
M13mp18/19, pUC18/19, pUC57, pTZ19R/U: 1 PhiX174, pBR322: 0
Digestion of Agarose-embedded DNA: A minimum of 5 units of KpnI is required for digestion of 1ug of agarose-embedded lambda DNA in 16 hours.
Methylation Effects
Dam, EcoBl, EcoKl: never overlaps - no effect Dcm, CpG: may overlap - no effect
Add
Nuclease free water: 16ul R1625-65: 2ul DNA (0.5-1ug/ul): 1ul KpnI: 0.5-2ul Mix gently, spin down for a few seconds. Incubate at 37ºC for 1-16 hours.
Add
PCR Reaction Mixture: 10ul (~0.1-0.5ug of DNA) Nuclease free water: 18ul R1625-65: 2ul KpnI 1-2ul Mix gently, spin down for a few seconds. Incubate at 37ºC for 1-16 hours.
Storage and Stability
May be stored at 4°C for short-term only. For long-term storage, store at -20°C. Aliquots are stable for 6 months after receipt at -20°C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.
Source
Klebsiella pneumoniae OK8
Important Note
This product as supplied is intended for research use only, not for use in human, therapeutic or diagnostic applications without the expressed written authorization of United States Biological.
