Restriction Enzymes

EcoRV

39,60 180,00 

EcoRV is a restriction enzyme isolated from E. coli J62plg 74 that cleaves the palindromic sequence GATATC.   

EcoRV creates blunt ends.

Concentration: 10U/µL

Source: Escherichia coli, J62 plg 74

Reagents supplied with: Buffer M and the universal Simple Buffer for double digests.

Store at: -20 °C

The Enzyme is supplied with 10x Buffer M (recommended) and the universal 10x Simple Buffer for double digests. The enzyme is also fully active in the respective buffers from other suppliers and can be used in double digests together with enzymes from other suppliers.

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Beschreibung

 

EcoRV

Activity in Simplebiotech buffers:

LMHSHSimple2x Simple
10-2510050<10100100

Rapid digest within 15 minutes.
10x Buffer M: 500 mM NaCl, 100 mM Tris-HCl (pH 7.9 at 25 °C), 100 mM MgCl2, 1000 μg/ml BSA
10x Simple buffer: 500 mM Potassium Acetate, 200 mM Tris-acetate, 100 mM Magnesium Acetate, 1000 μg/ml BSA, pH 7.9 at 25°C

10x Buffer M                   2 µL
DNA (0.5-1µg)                 1µL
10 U EcoRV                      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.

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.
Unit calculation assay conditions: 1 µg Lambda DNA has been digested in 100 mM Tris-HCl (pH 7.4 at 25 °C), 50mM NaCl, 5mM MgCl2, 0.025% Triton X-100 ,100 µg/mL BSA
Absence of contaminants: 50 units of EcoRV do not produce any unspecific cleavage products after 16 hrs incubation with 1µg of Lambda DNA at 37 °C. After 20-fold overdigestion with EcoRV, greater than 95% of the DNA fragments can be ligated and recut with this enzyme.
Storage buffer: EcoRV is supplied in 50 mM KCl, 10 mM Tris HCl (pH 7.4), 0.1 mM EDTA, 1 mM dithiothreitol, 200 μg/ml BSA and 50% glycerol.
Inactivation: Heat to 80 °C for 20 minutes. 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.
Methylation Sensitivity:
dam methylation: Not sensitive
dcm methylation: Not sensitive
CpG Methylation: Blocked by overlapping

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