Reaction & Storage Conditions Reaction Conditions: 1X NEBuffer 4 Supplemented with 100 μg/ml Bovine Serum Albumin Incubate at
37°C.
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 pBR322 DNA in 1 hour at 37°C in a total reaction volume of 50 µl.
Concentration: 4,000 units/ml
Unit Assay Substrate: pBR322 DNA
Storage Conditions: 20 mM Tris-HCl 500 mM KCl 1 mM MgCl2 0.1 mM EDTA 200 µg/ml BSA 50% Glycerol 0.1% Triton X-100
pH 7.0 @ 25°C
Storage Temperature: -20°C
Diluent Compatibility: Diluent B
Notes General notes:
KasI is an isoschizomer of NarI and SfoI.
KasI produces a 4-base 5´ extension whereas NarI produces a 2-base 5´ extension.
KasI demonstrates marked site preference and is 25-fold more active on λ DNA than on pBR322 DNA.
Usage notes:
-80°C is recommended for storage longer than 6 months.
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 20-fold overdigestion with KasI, > 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 KasI.
16-Hour Incubation:
A 50 μl reaction containing 1 μg of DNA
and 4 units of KasI 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 10 units of KasI with 1 μg of a mixture of single and double-stranded
[3H] E. coli DNA (105 cpm/μg) for 4 hours at 37ºC
released < 0.8% of the total radioactivity.
Blue/White Screening Assay:
An appropriate vector is digested at a unique site within the
lacZα gene with a 10-fold excess of enzyme.
The vector DNA is then ligated, transformed, and plated onto Xgal/IPTG/Amp plates.
Successful expression of β-galactosidase is a function of how intact its gene remains after cloning,
an intact gene gives rise to a blue colony, removal of even a single base gives rise to a white colony.
Enzyme preparations must produce fewer than 3% white colonies to be Blue/White certified.
