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Restriction Endonucleases >
Restriction Endonucleases >
KasI |
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Recognition Site:
isoschizomers | compatible ends | single letter code
Source:
A E. coli strain that carries the KasI gene from Kluyvera ascorbata (C. Polisson).
Reagents Supplied:
NEBuffer 4 (10X)
BSA (100X)
Enzyme Properties
Activity in NEBuffers:
| NEBuffer 1: | | 25% | | NEBuffer 2: | | 100% | | NEBuffer 3: | | 0% | | NEBuffer 4: | | 100% |
When using a buffer other than the optimal (supplied) NEBuffer, it may be necessary to add more enzyme to achieve complete digestion.
Methylation Sensitivity:
| dam methylation: Not sensitive | | dcm methylation: Not sensitive | | CpG methylation: Blocked |
Heat Inactivation:
65°C for 20 minutes
Survival in a Reaction:
Minimum units to digest 1 µg of substrate DNA in 16 hours: 1.00 unit(s)
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
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.
FAQs
- What isoschizomers are there?
- The NEB catalog has historically stated that activity in NEBuffer4 is less than 100% yet this enzyme is now supplied with NEBuffer4. What have you changed?
- Has the conversion to NEBuffer4 altered any of the properties of the restriction enzyme?
- If I have an old tube of enzyme, what NEBuffer should I use?
- Will the new enzyme work in the originally supplied NEBuffer?
- Why is NEB switching this restriction enzyme to NEBuffer 4?
- Are slow sites found in common vectors?
- Is KasI affected by methylation?
- Is extended digestion of KasI recommended?
- Are more units of KasI required to cut supercoiled DNA than lambda DNA?
- Is activity loss of KasI seen in 6 months or less?
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 30 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 (205 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.
Reagents Sold Separately
NEBuffer 4
BSA
Legal
Patents:
New England Biolabs, Inc.: U.S. Patent No. 5,147,794
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