Thermo Scientific™ PagI (BspHI)
The PagI (BspHI) restriction enzyme recognizes T^CATGA sites and cuts best at 37°C in O buffer (Isoschizomers: BspHI, CciI, RcaI).
Manufacturer: Thermo Scientific™ ER1281
Lambda DNA digested with PagI (BspHI), 0.7% agarose, 3 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'...T▵C A T G A...3'
3'...A G T A C▵T...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
- PagI is supplied in: 10mM Tris-HCl (pH 7.4 at 25°C), 100mM NaCl, 1mM DTT, 1mM EDTA, 0.2mg/mL BSA and 50% (v/v) glycerol
Ligation and Recleavage:
- After 50-fold overdigestion with PagI, more than 95% of the DNA fragments can be ligated and recut
- Dam: may overlap —cleavage impaired
- Dcm: never overlaps —no effect
- CpG: never overlaps —no effect
- EcoKI: never overlaps —no effect
- EcoBI: may overlap —cleavage impaired
Digestion of Agarose-embedded DNA:
- Minimum 5units of the enzyme are required for complete digestion of 1μg of agarose-embedded lambda DNA in 16 hours
- AflIII, PscI, DsaI, Eco130I, NcoI
Low salt, high glycerol (>5%) concentrations, pH >8.0 or a large excess of enzyme may result in star activity. PagI cleavage is impaired by overlapping dam methylation. To avoid dam methylation, use a dam-, dcm strain such as GM2163 (#M0099).