Restriction endonucleases

Description

Product Details

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 37^oC in a total reaction volume of 50 μl.

Digest Guidelines

• Standard reaction conditions: In order to overcome DNA source variability, a ~10 fold overdigest is suggested:

10U Kpn I	1μl
10x EQ buffer	5μl
DNA substrate	1μg
Sterile ultrapure water	Up to 50 μl

• Speed Digest reaction: Incubate for 15 minutes at 37^oC as in standard reaction in 1x KpnI buffer (10 mM Tris-HCl (pH 7.0 @ 25^oC), 10 mM MgCl2, 0.01% Triton X-100, 100 μg/ml BSA) or 1x eQ buffer (20 mM Tris-acetate (pH 7.9 @ 25^oC), 10 mM Mg-acetate, 50 mM K-acetate, 100 μg/ml BSA).

• Activity(%) in EnzyQuest buffers -Double digest tips

R1	R2	R3	R4	EQ
75-100	25-50	<10	<10	100

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

Methylation Sensitivity:

• 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 37^oC

Storage conditions:

• 50 mM KCl,
• 10 mM Tris-HCl (pH 7.4)
• 0.1 mM EDTA
• 1 mM dithiothreitol
• 200 μg/ml BSA
• 50% glycerol
• Store at -20^oC

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 37^oC.

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.