Enzima restrictiva de clasa III HpyCH4 III

Caracteristici standard:
  • Temperatura de incubare a enzimei este de 37 oC,iar daca enzima este supusa unei temperatura de 65 oC se inactiveaza.

  • Concentratia este de 5,000 unitati/ml, avand o dimensiunea este de 250 unitati.

  • Utilizeazand buferul CutSmart pentru a obtine o activitate ridicata.

  • Utilizand cantitatile corespunzatoare de ADN, componentele enzimatice si volumul de tampon corect se va obtine o digestie optima.

  • O unitate de enzima va digera complet 1 ug de substrat de ADN intr-o reactie de 50 pl in 60 de minute, iar pentru o activitate de 100% se recomanda utilizarea bufferului CutSmart realizand o digestie mai rapida.

Descriere

Actioneaza asupra fragmentului de ADN: 

                                5'...ACNGT...3'
                                3'...TGNCA...5'


ADN-ul ce este supus digestiei nu trebuie sa contina contaminati cum ar fi fenol, cloroform, alcool, EDTA, detergenti sau saruri in exces. Se recomanda etape de spalare suplimentare in timpul purificarii.

Metilarea ADN-ului poate inhiba digestia cu anumite enzime. 


Se pastreaza pe gheata cand nu se afla in congelator. Trebuie adaugata drept ultima componenta in reactie. In general, se recomanda 5-10 unitati de enzima per ug de ADN si 10-20 unitati pentru ADN genomic realizandu-se digestia intr-o ora. 

Specificatii complete
Product Name HpyCH4III
Concentration:  5,000 units/ml
Unit Definition: One unit is defined as the amount of enzyme required to digest 1 µg of Lambda DNA in 1 hour at 37°C in a total reaction volume of 50 µl.
Shelf Life: 24 months
Storage Temp: -20 °C
Storage Conditions: 100 mM NaCl, 10 mM Tris-HCl (pH 7.4), 1 mM DTT, 0.1 mM EDTA, 50% Glycerol, 200 µg/ml BSA
Specification Version: PS-R0618S/L v1.0
Effective Date: 23-Sep-13
Exonuclease Activity (Radioactivity Release) - A 50 µl reaction in CutSmart™ Buffer containing 1 µg of a mixture of single and doublestranded [ ³H] E. coli DNA and a minimum of 15 units of HpyCH4III incubated for 4 hours at 37ºC releases
Ligation and Recutting (Terminal Integrity) - After a 5-fold over-digestion of Lambda DNA with HpyCH4III, ~50% of the DNA fragments can be ligated with T4 DNA ligase in 16 hours at 16ºC. Of these ligated fragments, >95% can be recut with HpyCH4III.
Non-Specific DNase Activity (16 Hour) ) - A 50 µl reaction in CutSmart™ Buffer containing 1 µg of Lambda DNA and a minimum of 5 units of HpyCH4III incubated for 16 hours at 37ºC results in a DNA pattern free of detectable nuclease degradation as determined by agarose gel electrophoresis.