Unit definition: One unit is defined as amount of enzyme required to produce a complete digest of 1 µg Lambda DNA in 60 min in a total reaction volume of 50µL under the assay conditions.
Unit substrate: Lambda DNA/EcoRI digest
Unit calculation assay conditions: 10 mM Tris-HCl (pH 7.0 @ 25 °C), 10 mM MgCl2, 1 mM dithiothreitol, 0.01% Triton X-100, 100 μg/ml bovine serum albumin and DNA. Incubate at 37 °C
Absence of contaminants: 10 units of KpnI do not produce any unspecific cleavage products after 16 hrs incubation with 1µg of Lambda/EcoRI DNA at 37 °C. After 10-fold overdigestion with KpnI, greater than 95% of the DNA fragments can be ligated and recleaved with this enzyme.
Storage buffer: KpnI is supplied in 10 mM Tris HCl (pH 7.4), 0.1 mM EDTA, 1 mM DTT, 200 μg/ml BSA and 50% glycerol.
Inactivation: KpnI cannot be heat inactivated in 20min at 80 °C. The enzyme can be removed by spin column purification. It is also possible to stop the digest by addition of EDTA to a final concentration of >10mM or SDS to a concentration of 0.1 %. Alternatively use our environmentally friendly 6x orange gel loading buffer. For further enzymatic reactions remove SDS or EDTA by spin column-based PCR purification kit.
Star activity: Conditions of low ionic strength, high enzyme concentration, glycerol concentration >5% or pH >8.0 may result in star activity.
dam methylation: Not sensitive
dcm methylation: Not sensitive
CpG Methylation: Not sensitive
Activity in Simplebiotech buffers:
*May exhibit star activity under these conditions.
10x UKpnI Buffer: 100 mM Tris-HCl (pH 7.0 @ 25 °C), 100 mM MgCl2, 0.1% Triton X-100, 100 mg/ml bovine serum albumin.
10x Simple buffer: 500 mM Potassium-acetate, 200 mM Tris-acetate (pH 7.9 at 25°C), 100 mM Magnesium Acetate, 1000 μg/ml BSA
General reaction mixture:
10x buffer UKpnI 2 µL
DNA (0.5-1µg) 1µL
10 U KpnI 1 µL
Nuclease free water 16 µL
Final volume 20µL
Incubate at 37 °C for 15 minutes.
Always add the restriction enzymes as last component. Mix gently but thoroughly by pipetting up and down or flicking. Spin down.
Incubate at 37 °C for 15min. Restriction enzymes are stored in 50% glycerol. The glycerol concentration in the reaction mix should not exceed 10%. When digesting more than 1µg DNA, scale up the restriction mix or prolong the incubation time.