KpnI is a restriction enzyme purified from Klebsiella pneumonia OK8.
Reagents supplied: 10x KpnI and 10x EQ buffer
Unit definition: One unit is the amount of enzyme required to digest 1 μg of Lambda DNA (EcoR I digest) in 60 min at 37oC in a total reaction volume of 50 μl.
Standard reaction conditions: In order to overcome DNA source variability, a ~10 fold overdigest is suggested:
10U Kpn I
10x EQ buffer
Sterile ultrapure water
Up to 50 μl
Speed Digest reaction: Incubate for 15 minutes at 37oC as in standard reaction in 1x KpnI buffer (10 mM Tris-HCl (pH 7.0 @ 25oC), 10 mM MgCl2, 0.01% Triton X-100, 100 μg/ml BSA) or 1x eQ buffer (20 mM Tris-acetate (pH 7.9 @ 25oC), 10 mM Mg-acetate, 50 mM K-acetate, 100 μg/ml BSA).
Activity(%) in EnzyQuest buffers-Double digest tips
Reactions were performed as for unit definition reaction. The above data could be used as guideline for double digest reactions. Our enzymes are also active and compatible in buffer systems fromRE020S, RE020L, REc020S, REc020L other vendors.
Heat inactivation: No
dam methylation: Not sensitive
dcm methylation: Not sensitive
CpG methylation: Not sensitive
Unit calculation assay conditions:
1x KpnI buffer
Lambda DNA (EcoRI digest)
Incubation at 37oC
50 mM KCl,
10 mM Tris-HCl (pH 7.4)
0.1 mM EDTA
1 mM dithiothreitol
200 μg/ml BSA
Store at -20oC
Non-specific nuclease activity: 30 units of Kpn I do not produce any unspecific cleavage products after 16 hrs incubation with 1 μg of Lambda DNA (EcoR I digest) at 37oC.
Re-ligation and recut: After 10-fold overdigestion with Kpn I, greater than 95% of the DNA fragments can be ligated and recut with this enzyme.
Star activity: Conditions of low ionic strength, high enzyme concentration, glycerol concentration >5% or pH >8.0 may result in star activity.
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