1X NEBuffer 4: 20 mM Tris-acetate 50 mM potassium acetate 10 mM Magnesium Acetate 1 mM Dithiothreitol
pH 7.9 @ 25°C
Unit Definition: One unit is defined as the amount of enzyme required to digest 1 µg of λ DNA in 1 hour at 37°C in a total reaction volume of 50 µl.
Concentration: 10,000 units/ml
Unit Assay Substrate: λ DNA
Storage Conditions: 10 mM Tris-HCl 50 mM KCl 1 mM Dithiothreitol 0.1 mM EDTA 200 µg/ml BSA 50% Glycerol
pH 7.4 @ 25°C
Storage Temperature: -20°C
Diluent Compatibility: Diluent A
Notes General notes:
HinP1I is an isoschizomer of HhaI.
HinP1I produces a 5´ extension, whereas HhaI produces a 3´ extension. The 5´ extension can be efficiently ligated into the AccI site of M13 and pUC cloning vectors.
Quality Control for Current Lot Quality control values for a specific lot can be found on the datacard which accompanies each product.
Ligation and Re-cutting:
After a 10-fold overdigestion with HinP1I, > 95%
of the DNA fragments can be ligated with T4 DNA Ligase (at a 5' termini concentration of 1-2 μM)
at 16ºC. Of these ligated fragments, > 95% can be recut with HinP1I.
16-Hour Incubation:
A 50 μl reaction containing 1 μg of DNA
and 100 units of HinP1I incubated for 16 hours at 37ºC
resulted in a DNA pattern free of detectable nuclease degradation as determined by agarose gel electrophoresis.
Exonuclease Activity:
Incubation of a 50 μl reaction containing 100 units of HinP1I with 1 μg of a mixture of single and double-stranded
[3H] E. coli DNA (205 cpm/μg) for 4 hours at 37ºC
released < 0.1% of the total radioactivity.
