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Cat. No.: ISCEI-250 (for 250U)

Recognition Site

TAGGGATAACAGGGTAAT
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'

Description

I-SceI is a type II intron-encoded endonuclease derived from Saccharomyces cerevisiae (baker's yeast). It specifically recognizes and cleaves an 18-base pair non-palindromic DNA sequence (5'-TAGGGATAACAGGGTAAT-3'), generating a 4-base 3' overhang sticky end. Due to its exceptionally long recognition sequence, the natural occurrence of this site in the genome is extremely rare (approximately once every 7×10¹⁰ random base pairs). This makes I-SceI an ideal tool for precise genome manipulation.​

Components

• I-SceI (5 U/μl): 50 μl
• pUC-I-SceI (100 ng/μl): 20 μl
• 10× Buffer: 1 ml
• 10×  Color Buffer: 1 ml

Activity Definition

One unit (U) of activity is defined as the amount of enzyme required to completely digest 1 μg of pUC-ISceI DNA at 37°C within 1 hour.​

Application

• High-resolution m6A methylation analysis;
• mRNA drug sequence analysis.

Quality Control

• Functional Activity Assay​: At 37°C, in a 20 μl universal reaction system, 1 μl of I-SceI can completely digest 1 μg of pUC-I-SceI within 15 minutes.
• ​Extended Incubation Test (Overlong Digestion)​​: At 37°C, in a 20 μl universal reaction system, 1 μl of I-SceI was co-incubated with 1 μg of pUC-I-SceI for 3 hours. No nonspecific degradation of the substrate caused by other nucleases (indicating absence of contamination) was detected.
• ​Digestion-Ligation-Redigestion Assay​: At 37°C, DNA substrate was digested with 10-fold molar excess of I-SceI. The digested products were recovered, and over 95% of them could be re-ligated at 22°C using T4 DNA Ligase (Fast). After recovering the ligated products, approximately 95% or more of them could be re-cut by the same endonuclease.

Storage

-20°C.

Precautions

• ​Recognition Sequence Specificity​: Homing endonucleases, unlike restriction endonucleases, do not have strictly defined recognition sequences. Therefore, single-base mutations do not abolish their recognition function but may reduce cleavage efficiency to varying degrees. The exact regions of essential bases within the recognition sequence remain undetermined. The listed recognition sequences are experimentally confirmed sites that are known to be recognized and cleaved.
• ​Control Template and Applications​: The enzyme is provided with control plasmid DNA at a concentration of 100 ng/μl. Digestion with I-SceI generates a 2711 bp fragment. The pUC-I-SceI plasmid carries ampicillin resistance and can be transformed and amplified by users for additional experiments.