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: 100 mM NaCl, 50 mM Tris-HCl (pH 7.9 at 25°C), 10 mM MgCl2, 1mM DTT, 0,02% Triton X-100, 100 µg/mL bovine serum albumin.
Absence of contaminants: 60 units of NcoI incubated for 16 hours at 37 °C with 1 μg of Lambda DNA do not produce any unspecific cleavage products. After 10-fold overdigestion with NcoI, greater than 95% of the DNA fragments can be ligated and recut with this enzyme.
Storage buffer: NcoI is supplied in 50 mM KCl, 10 mM TrisHCl (pH 7.4), 0.1 mM EDTA, 1 mM dithiothreitol, 200 μg/ml BSA and 50% glycerol.
Inactivation: Heat to 65 °C for 20 minutes. Inactivation is also possible 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: Not sensitive
Star activity: High enzyme concentration or glycerol concentration >5% may result in star activity.
Activity in Simplebiotech buffers:
L M H SH Simple
50-75 75-100 100 100 100
10x Simple buffer: 200 mM Tris acetate (pH 7.9 at 25 °C), 100 Mg acetate 500mM K acetat, 1mg/mL BSA
10x Simple buffer 2 µL
DNA (0.5-1µg) 1µL
10 U NcoI 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.