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Homing Enzyme: I-SceI

5'-T A G G G A T A A^C A G G G T A A T-3' 3'-A T C C C^T A T T G T C C C A T T A-5'		digested pUC-I-SceI DNA 1.4% agarose
#ER1771 Supplied with: 10X Buffer Tango™ 10X Buffer Tango™ without Mg-acetate 100 mM Mg-acetate	250 u   1 ml 1 ml 1 ml
Related Documents (in pdf, ~55 KB): Certificate of Analysis: #ER1771 MSDS (English) MSDS (English-USA) MSDS (German) Chart (in pdf, 721 KB)

Description
I-SceI is a site-specific homing endonuclease encoded by a mitochondrial intron of Saccharomyces cerevisiae (1, 2). Intron-encoded endonucleases are proteins that promote the first step in mobility of the intron at the DNA level. They recognize and cleave an intronless allele of their cognate gene to insert a copy of the intron by a double-strand-break repair mechanism that results in the recipient allele also becoming intron-plus (3-5). Homing endonucleases recognize long, 14-40 base pairs sequences and are, therefore, extremely rare-cutting restriction enzymes. They allow the introduction of a single or several double-strand breaks into complex genomes. This capability makes the enzymes powerful tools in high-resolution physical mapping, genome organization analysis, gene cloning and site-directed induced-recombination and for studying double-strand-break repair in diverse biological systems (4, 6).

Concentration
10 u/µl

Conditions for 100% Activity

Recommended buffer	Incubation temperature	Units for  overnight  incubation, u/µg DNA	Thermal inactivation, in 20 min	Enzyme activity, %
				B  (blue)	G  (green)	O  (orange)	R  (red)	Tango™  (yellow)
				1X	1X	1X	1X	1X	2X
Buffer Tango™	37°C	0.5	65°C	50-100	50-100	50-100	50-100	100	50-100

Storage Buffer
10 mM Tris-HCl (pH 7.4 at 25°C), 500 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 50% glycerol.

Ligation and Recutting
After 50-fold overdigestion with I-SceI, more than 95% of the digested DNA fragments can be ligated and recut.

Methylation Effects
Dam: Dcm: CpG: EcoKI: EcoBI:	never overlaps  never overlaps  never overlaps  never overlaps  never overlaps	- no effect. - no effect. - no efect. - no effect. - no effect.

Digestion of Agarose-embedded DNA
Minimum of 20 units of the enzyme are required for complete digestion of 1 µg of agarose-embedded pUC-I-SceI DNA in 1 hour (see the protocol below).

Note

• Homing endonucleases do not have stringently defined recognition sequences. They can tolerate minor sequence changes, which only partially affect the cleavage reaction.
• I-SceI may remain associated with the cleaved DNA. This may cause DNA band shifting during electrophoresis. To avoid an atypical DNA band pattern, use the 6X Loading Dye & SDS Solution for sample preparation or heat the digested DNA in the presence of SDS prior to electrophoresis.
• Diffusion of the enzyme in the absence of Mg-acetate prior to digestion is necessary, because I-SceI is unstable in the presence of Mg²⁺ ions.
• Assayed using pUC-I-SceI DNA.

Double Digestions using Tango™ Buffer, DoubleDigest™

Optimal buffer	Enzyme activity, %
	Tango™  (yellow)
	1X	2X
Buffer Tango™	100	50-100

This product is licensed under one or more U.S. Patents No. US5,474,896, US5,866,361, US 5 792 632 or corresponding foreign patents.
Certain countries are out of the scope of patent coverage.

Protocol for Digestion of the Agarose-embedded DNA with I-SceI

1. Immerse an agarose plug in 50-100 µl of the 1X Tango™ buffer without Mg-acetate (supplied with the enzyme). The volume of the buffer should be sufficient to completely cover the plug.
2. Add 20 u of the enzyme.
3. Incubate 2 hours on ice.
4. Add 1/10 volume of the 100 mM Mg-acetate solution (supplied with the enzyme).
5. Incubate at 37°C for 1 hour.

References

1. Colleaux, L., et al., Recognition and cleavage site of the intron-encoded omega transposase, Proc. Natl. Acad.Sci. U.S.A.,85, 6022-6026, 1988.

2. Monteihet, C., et al., Purification and characterization of the in vitro activity of I-SceI, a novel and highly specific endonuclease encoded by a group I intron, Nucleic Acids Res., 18, 1407-1413, 1990.
3. Dujon, B., Group I introns as mobile genetic elements: facts and mechanistic speculations - review, Gene, 82, 91-114, 1989.
4. Belfort, M., Roberts R.J., Homing endonucleases: keeping the house in order, Nucleic Acids Res., 25, 3379-3388, 1997.
5. Chevalier, B.S., Stoddard, B.L., Homing endonucleases:structural and functional insight into the catalysis of intron/intein mobility, Nucleic Acids Res., 29, 3757-3774, 2001.
6. Jasin, M., Genetic manipulation of genomes with rare-cutting endonucleases, Trends in Genetics, 12, 224-228, 1996.

Updated vasario 06, 2008 15:57