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Restriction Endonucleases >
Homing Endonucleases >
I-SceI |
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Prices are in US dollars and valid only for US orders.
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Recognition Site:
isoschizomers | compatible ends | single letter code
Description:
I-SceI is an intron-encoded endonuclease. The intron encoding I-SceI is present in the mitochondria of Saccharomyces cerevisiae.
Source:
A E. coli strain that carries the I-SceI mitochondrial gene from Saccharomyces cerevisiae (B. Dujon).
Reagents Supplied:
NEBuffer I-SceI (10X)
BSA (100X)
pGPS2 NotI-linearized Control Plasmid (5 μg)
Enzyme Properties
Activity in NEBuffers:
| NEBuffer 1: | | 10% | | NEBuffer 2: | | 50% | | NEBuffer 3: | | 50% | | NEBuffer 4: | | 50% |
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 I-SceI
Supplemented with 100 μg/ml Bovine Serum Albumin
Incubate at
37°C.
1X NEBuffer I-SceI:
10 mM Tris-HCl
10 mM MgCl2
1 mM Dithiothreitol
pH 8.8 @ 25°C
Unit Definition:
One unit is defined as the amount of enzyme required to cleave 1 µg of pGPS2 NotI-Iinearized Control Plasmid in 1 hour at 37°C in a total reaction volume of 50 µl.
Concentration:
5,000 units/ml
Unit Assay Substrate:
pGPS2 NotI-linearized Control Plasmid
Storage Conditions:
10 mM Tris-HCl
300 mM NaCl
1 mM Dithiothreitol
0.1 mM EDTA
500 µg/ml BSA
50% Glycerol
pH 7.4 @ 25°C
Storage Temperature:
-80°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. NotI-linearized pGPS2 is supplied at 0.5 mg/ml in 10 mM Tris-HCl (pH 8.0), 1 mM EDTA. Cleavage of this 2499 bp plasmid with I-SceI gives fragments of 1518 and 981 base pairs.
- Storage: Using this product to study transgenesis requires the enzyme to be stored at -80°C. For simple DNA digestions, this product can be stored at -20°C. See the following reference for more information: Rembold, M. et. al. (2006) Nature Protocols 1, 1133-1139.
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 I-SceI, > 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-SceI.
Cleavage by I-SceI leaves DNA fragments with four nucleotide 3' extensions. Fragments with complimentary ends can be joined by T4 DNA Ligase.
16-Hour Incubation:
A 50 μl reaction containing 1 μg of DNA
and 25 units of I-SceI 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-SceI 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 5 units of I-SceI 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:
pGPS2 NotI-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 2,499 bp plasmid with I-SceI gives fragments of 1,518 and 981 base pairs.
Reagents Sold Separately
NEBuffer I-SceI
BSA
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