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Restriction Endonucleases >
Homing Endonucleases >
I-CeuI |
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Recognition Site:
isoschizomers | compatible ends | single letter code
Description:
I-CeuI is an intron-encoded endonuclease. The intron encoding I-CeuI is present in the chloroplast large rRNA gene of Chlamydomonas eugametos (1). This gene has been cloned and overexpressed in E. coli (2).
Source:
A E. coli strain that carries the I-CeuI gene from Chlamydomonas eugametos (C. Lemieux).
Reagents Supplied:
NEBuffer 4 (10X)
BSA (100X)
pBHS ScaI-linearized Control Plasmid (5 μg)
Enzyme Properties
Activity in NEBuffers:
| NEBuffer 1: | | 10% | | NEBuffer 2: | | 10% | | 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.
Heat Inactivation:
65°C for 20 minutes
Survival in a Reaction:
Minimum units to digest 1 µg of substrate DNA in 16 hours: 0.25 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 cleave 1 µg of pBHS ScaI-linearized Control Plasmid in 3 hours at 37°C in a total reaction volume of 50 µl.
Concentration:
5,000 units/ml
Unit Assay Substrate:
pBHS ScaI-linearized Control Plasmid
Storage Conditions:
10 mM Tris-HCl
200 mM KCl
1 mM Dithiothreitol
0.1 mM EDTA
200 µg/ml BSA
50% Glycerol
pH 8.0 @ 25°C
Storage Temperature:
-20°C
Diluent Compatibility:
Diluent B
Notes
General notes:- Homing endonucleases do not have stringently-defined recognition sequences in the way that restriction enzymes do. That is, single base changes do not abolish cleavage but reduce its efficiency to variable extents. The precise boundary of required bases is generally not known. The recognition sequence listed is one site that is known to be recognized and cleaved.
- Supplied with plasmid DNA. ScaI-linearized pBHS is supplied at 0.5 mg/ml in 10 mM Tris-HCl (pH 8.0), 1 mM EDTA. Cleavage of this 3.5 kb plasmid gives fragments of 2100 and 1400 base pairs.
Usage notes:- Digests should be incubated in 0.5% SDS prior to electrophoresis.
FAQs
- Can a double digest be performed with PI-SceI and I-CeuI?
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 I-CeuI, > 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 I-CeuI.
16-Hour Incubation:
A 50 μl reaction containing 1 μg of DNA
and 50 units of I-CeuI 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 50 units of I-CeuI 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.
Endonuclease Activity:
Incubation of a 50 μl reaction containing 50 units of I-CeuI with 1 μg of
ΦX174 RF I DNA for 4 hours at 37ºC resulted
in < 10% conversion to RFII as determined by agarose gel electrophoresis.
Plasmid DNA:
pBHS ScaI-linearized Control Plasmid is supplied at 0.5 mg/ml in 10 mM Tris-HCl (pH 8.0) and 1 mm EDTA. Cleavage of this 3.5 kb plasmid gives fragments of 2,100 and 1,400 base pairs.
Reagents Sold Separately
NEBuffer 4
BSA
Legal
Patents:
New England BioLabs, Inc.: U.S. 5,420,032
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