HindIII is a restriction enzyme is a restriction enzyme purified from a recombinant E. coli that carries a plasmid encoding the HindIII gene from Haemophilus influenzae. HindIII cleaves the palindromic sequence A▼AGCTT.
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 50mM NaCl, 10 mM Tris-HCl (pH 7.9 at 25 °C), 10 mM MgCl2, 100 µg/mL BSA
Absence of contaminants: 50 units of HindIII do not produce any unspecific cleavage products after 16 hrs incubation with 1µg of Lambda DNA at 37 °C. After 100-fold overdigestion with HindIII, greater than 98% of the DNA fragments can be ligated and recut with this enzyme.
Storage buffer: HindIII is supplied in 50 mM KCl, 10 mM Tris HCl (pH 7.4), 0.1 mM EDTA, 1 mM dithiothreitol, 500 μg/ml BSA and 50% glycerol.
Inactivation: Heat to 65 °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: 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.
10x Buffer M 2 µL
DNA (0.5-1µg) 1µL
10 U HindIII 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.