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Conventional Restriction Enzymes

I-SceI

Product information

Reaction conditions

Recommended buffer for 100% activity	Optimal tempThermo Scientificure	Enzyme activity in Thermo Scieitnfic buffers, %						Tango™ buffer for double digestion
		B (blue) 1X	G (green) 1X	O (orange) 1X	R (red) 1X	Tango™ (yellow) 1X / 2X
Tango™	37°C	50-100	50-100	50-100	50-100	100	50-100	1X or 2X

pUC-I-Scel DNA
1.4% agarose

Conditions for 100% Activity

1X Buffer Tango™:
33 mM Tris-acetate (pH 7.9 at 37°C), 10 mM Mg-acetate, 66 mM K-acetate and 0.1 mg/ml BSA.
Incubate at 37°C.

Storage Buffer

I-SceI is supplied in:
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 Recleavage

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

Digestion of Agarose-embedded DNA

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

Note

• Homing enzymes 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 atypical DNA band patterns, use the 6X DNA Loading Dye & SDS Solution for sample preparation or heat the digested DNA in the presence of SDS prior to electrophoresis.
• Assayed using pUC-I-SceI DNA.

Isoschizomers

Search for commercial isoschizomers using REsearch™.

Double Digestion

Perform double digestion using DoubleDigest™.

Methylation Effects

Dam: never overlaps – no effect.
Dcm: never overlaps – no effect.
CpG: never overlaps – no effect.
EcoKI: never overlaps – no effect.
EcoBI: never overlaps – no effect.

Cleavage efficiency close to the termini of PCR fragments

bp from the recognition site to fragment end
1	2	3	4	5
50-100

Patents, Licenses, Trademarks

Protocols & recommendations

RECOMMENDATIONS FOR USE

ADDITIONAL PROTOCOLS

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.

Note

* 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.

DNA Digestion

We recommend digesting 0.2-1.5 µg DNA with a 2-fold to 10-fold excess of enzyme in a total volume of 20 µl. A typical restriction enzyme digestion protocol is below.

1. Add the following reaction components in the order indicated:

   Water, nuclease-free	16-16.5 µl
   10X recommended buffer for restriction enzyme	2 µl
   Substrate DNA	1 µl (~1 µg)
   Restriction enzyme	0.5-1 µl (5-10 u)
   Total volume	20 µl

2. Mix gently and spin down briefly.
3. Incubate at the optimal reaction temperature for 1-16 hours.

Note

• The digestion reaction may be scaled either up or down.
• Some enzymes require additional components to obtain the stated activity. In these cases, add the required additive and adjust the volume of water appropriately.

Digestion of PCR Products

The most convenient option for digestion of PCR-amplified DNA is the addition of a restriction enzyme directly to the reaction tube after completion of PCR. The majority of Fermentas restriction enzymes are active in Fermentas PCR buffers.
However, digestion of PCR products in the amplification mixture is often inefficient. Therefore, PCR reaction mixture should not make more than 1/3 volume of digestion reaction mixture to avoid inhibitory effects.

1. Add the following reaction components in the order indicated:

   PCR reaction mixture	10 µl (~0.1-0.5 µg of DNA)
   Water, nuclease-free	16-17 µl
   10X recommended buffer for restriction enzyme	2 µl*
   Restriction enzyme	1-2 µl (10-20 u)
   Total volume	30 µl

   * Only 2 µl of 10X reaction buffer is required for unpurified PCR product in a 30 µl reaction volume.
2. Mix gently and spin down briefly.
3. Incubate at the optimal reaction temperature for 1-16 hours.

Note

• For cloning applications, purification of PCR products prior to digestion is necessary to remove the active thermophilic DNA polymerase present in the PCR mixture. DNA polymerases may alter the ends of the cleaved DNA and reduce the yield of ligation.
• If the restriction enzyme requires special additives (e.g., SAM), reduce the amount of water appropriately.
• If cleavage of the PCR product is inefficient purify the PCR products with the GeneJET™ PCR Purification Kit (#K0702) prior to digestion.
• After digestion, gel-purify the PCR product with the GeneJET™ Gel Extraction Kit (#K0692) or Silica Bead DNA Gel Extraction Kit (#K0513) to remove short DNA fragments which compete with the insert in a ligation reaction.

Double Digestion

DoubleDigest™ Engine
Our DoubleDigest™ engine is a great tool to find information on buffer and reaction conditions for your double digests. Simply select two restriction enzymes required for digestion, submit the query and follow the recommendations. The DoubleDigest™ engine is continuously updated with newly released Fermentas restriction enzymes.

Digestion of Agarose-embedded DNA

1. Embed the substrate DNA in 1% low melting temperature agarose, 1 µg / 30 µl.
2. Prepare ~30 µl agarose plugs with a GelSyringe® or similar agarose dispensing system.
3. Equilibrate the plug in 100 µl of the appropriate 1X restriction enzyme buffer for 15 min.
4. Place the plug in 100 µl of fresh 1X buffer containing the restriction enzyme (see Table "Digestion of Agarose-embedded DNA").
5. Incubate at 30-37°C for mesophilic enzymes or at 50-55°C for thermophilic enzymes for 4-16 hours.

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.