Thermo Scientific™ NotI
The NotI restriction enzyme recognizes GC^GGCCGC sites and cuts best at 37°C in O buffer (Isoschizomers: CciNI).
Manufacturer: Thermo Scientific™ ER0592
Ad2 DNA digested with NotI, 0.7% agarose, 7 cleavage sites
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.
5'..G C▵G G C C G C...3'
3'..C G C C G G▵C G...5'
Conditions for 100% Activity:
- 1X Buffer O: 50mM Tris-HCl (pH 7.5 at 37°C), 10mM MgCl2, 100mM NaCl and 0.1mg/mL BSA
- Incubate at 37°C
- NotI is supplied in: 20mM Tris-HCl (pH 7.8 at 25°C), 100mM NaCl, 0.1mM EDTA, 1mM DTT, 0.02% Triton X-100, 0.2mg/mL BSA and 50% (v/v) glycerol
Ligation and Recleavage:
- After 50-fold overdigestion with NotI, more than 95% of DNA fragments can be ligated and recut
- Dam: never overlaps —no effect
- Dcm: never overlaps —no effect
- CpG: completely overlaps —blocked
- EcoKI: never overlaps —no effect
- EcoBI: never overlaps —no effect
Digestion of Agarose-embedded DNA:
- Minimum 5units of the enzyme are required for complete digestion of 1μg of agarose-embedded Adenovirus-2 DNA in 16 hours
- Bsp120I, CfrI, Eco52I
Assayed using pTZ19RJL2 plasmid DNA digested with BseLI. Supercoiled plasmids may require up to 5-fold more NotI for complete digestion than linear DNAs.