Restriction endonucleases

Description

Product Details

EcoRV is a restriction enzyme purified from E. coli J62plg 74.

Reagents supplied: 10x R2 and 10x EQ buffer

Unit definition: One unit is the amount of enzyme required to digest 1 μg of Lambda DNA 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 EcoR V	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 R2 buffer (50 mM NaCl, 10 mM Tris-HCl (pH 7.9 @ 25^oC), 10 mM MgCl2, 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
10-25	100	50-75	<10	75-100

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 from other vendors.

Heat inactivation: 80^oC for 20 minutes

Methylation Sensitivity:

• dam methylation: Not sensitive
• dcm methylation: Not sensitive
• CpG methylation: Blocked by overlapping

Unit calculation assay conditions:

• 1x R2 buffer
• Lambda DNA
• 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: 100 units of EcoR V do not produce any unspecific cleavage products after 16 hrs incubation with 1 μg of Lambda DNA at 37^oC.

Re-ligation and recut: After 20-fold overdigestion with EcoR V, 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.