Activity in Simplebiotech buffers:
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.
dam methylation: Not sensitive
dcm methylation: Not sensitive
CpG Methylation: Blocked by overlapping