I-SceI

Detailní popis

Thermo Scientific conventional restriction endonucleases are a large collection of high quality restriction enzymes, optimized to work in one of the buffers of the Five Buffer System. In addition, the universal Tango buffer is provided for convenience in double digestions. All of the enzymes exhibit 100% activity in the recommended buffer and reaction conditions. To ensure consistent performance, Thermo Scientific restriction enzyme reaction buffers contain premixed BSA, which enhances the stability of many enzymes and binds contaminants that may be present in DNA preparations.

Highlights

• Superior quality – stringent quality control and industry leading manufacturing process
• Convenient color-coded Five Buffer System
• Includes universal Tango buffer for double-digestions
• BSA premixed in reaction buffers
• Wide selection of restriction endonuclease specificities

Applications

• Molecular cloning
• Restriction site mapping
• Genotyping
• Southern Blot
• Restriction fragment length polymorphism (RFLP)
• SNP

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.

  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
  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).
  Double Digestion	Perform double digestion using DoubleDigest.
  Hazardous	No
  Isoschizomers	Search for commercial isoschizomers using REsearch.
  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.
  Storage Condition	-20 C

  Reaction conditions

  Recommended buffer for 100% activity	Optimal temperature	Enzyme activity in Thermo Scientific 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

  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