Restriction Enzymes


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KpnI is a restriction enzyme purified from Klebsiella pneumonia OK8 that cleaves the palindromic sequence GGTAC▼C.

Concentration: 10U/µL
Source: Klebsiella pneumonia OK8.
Reagents supplied with: Buffer 10x UKpnI and 10x Simple Buffer.
The Enzyme is also active in respective buffers from other suppliers and can combined with enzymes from other suppliers in double digests.
Store at: -20 °C

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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.
Methylation Sensitivity:
dam methylation: Not sensitive
dcm methylation: Not sensitive
CpG Methylation: Not sensitive

Activity in Simplebiotech buffers:

L M H SH Simple 2x Simple
75-100* 25-50 <10 <10 100 50

*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.


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