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You are here:Home » Molecular Biology » MB-Enzymes, Restriction » KpnI

KpnI

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Specifications

5'-G G T A C^C-3'
3'-C^C A T G G-5'
Catalog #K1894
SourceKlebsiella pneumoniae OK8
Concentration10u/ul
Unit DefinitionOne 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 WithR1625: 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, .02% Triton X-100 and 0.1mg/ml BSA. Incubate at 37°C.
Dilution Buffer10mM 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 InactivationKpnI is inactivated by incubation at 80°C for 20min.
CAS NumberN/A
Overdigestion AssayNo 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) AssayThe 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 IncubationA minimum of 0.2 units of KpnI is required for complete digestion of 1ug of lambda DNA in 16 hours at 37°C.
Number of Recognition Sites in DNA
Lambda2:
M13mp18/19, pUC18/19, pUC57, pTZ19R/U1
PhiX174, pBR3220
Digestion of Agarose-embedded DNAA minimum of 5 units of KpnI is required for digestion of 1ug of agarose-embedded lambda DNA in 16 hours.
Methylation Effects
Dam, EcoKlNever overlaps - no effect Dcm, CpG: may overlap - no effect
EcoBlMay overlap - cleavage impaired.
Recommended Protocol for Digestion
Add
Nuclease free water16ul
R1625-652ul
DNA (0.5-1ug/ul)1ul
KpnI0.5-2ul
Mix gently, spin down for a few seconds.
Incubate at 37ºC for 1-16 hours.
Protocol for Digestion of PCR products directly after amplification
Add
PCR Reaction Mixture10ul (~0.1-0.5ug of DNA)
Nuclease free water18ul
R1625-652ul
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 12 months at -20°C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.


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