NheI

Beschreibung

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/HindIII digest
Unit calculation assay conditions: 50 mM potassium acetate, 20 mM Tris-acetate (pH 7.9 @ 25°C), 10 mM magnesium acetate, 100 μg/ml BSA. Incubation at 37 °C
Absence of contaminants: 40 units of NheI do not produce any unspecific cleavage products after 16 hrs incubation with 1µg of Lambda/HindIII DNA at 37 °C. After 50-fold overdigestion with NheI, greater than 98% of the DNA fragments can be ligated and recleaved with this enzyme.
Storage buffer: NheI is supplied in 200 mM NaCl, 10 mM Tris HCl (pH 7.4), 0.1 mM EDTA, 1 mM DTT, 0.15% Triton X-100, 200 μg/ml BSA and 50% glycerol.
Inactivation: NheI can be heat inactivated in 20min at 65 °C. The enzyme can be removed by spin column purification. 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.
Note: NheI cleved sites leave a 5’ CTAG extension, which can be efficiently ligated to DNA fragments generated by SpeI, XbaI or AvrII. The newly created junction cannot be recleaved with any of these enzymes.
Methylation Sensitivity:
dam methylation: Not sensitive
dcm methylation: Not sensitive
CpG Methylation: Not sensitive

Activity in Simplebiotech buffers:

The Enzyme is supplied with 10x buffer M and the 10x universal Simple Buffer for double digests. The enzyme is also fully active in the respective buffers from other suppliers and may be used in double digests together with enzymes from other suppliers.
10x Buffer M: 500mM NaCl, 100mM Tris-HCl (pH 7.9 at 25 °C), 100mM MgCl2, 1mg/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

Rapid digest within 15 minutes. 

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

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

Rapid digest within 15 minutes.

10xSimple Buffer                  2 µL

DNA (0.5-1µg)                     1µL

10 U SalI                            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.