Enzima restrictiva de clasa III Afl III

Caracteristici standard:
  • Enzima provine dintr-o tulpina de E.coli care poarta gena Afl II clonata din Anabaena flos-aquae. (CCAP 1403/13f).

  • Temperatura de incubare a enzimei este de 37 oC,iar daca este supusa unei temperaturi de 80 oC enzima se inactiveaza.

  • Concentratia este de 10,000 unitati/ml, iar dimensiunea este de 250 unitati.

  • Provine dintr-o tulpina de E.coli care poarta gena Afl II clonata din Anabaena flos-aquae. (CCAP 1403/13f).

  • Utilizand cantitatile corespunzatoare de ADN, componentele enzimatice si volumul de tampon corect se 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.

Descriere

Actioneaza asupra fragmentului de ADN: 

                                5'...ACRYGT...3'
                                3'...TGYRCA...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 realizand digestia intr-o ora. 

Specificatii complete
Product Name AflIII
Concentration:  10,000 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: 500 mM NaCl , 10 mM Tris-HCl (7.4), 1 mM DTT , 0.1 mM EDTA , 50 % Glycerol , 200 µg/ml BSA
Specification Version: PS-R0541S/L v1.0
Effective Date: 01-May-13
Exonuclease Activity (Radioactivity Release) - A 50 µl reaction in NEBuffer 3.1 containing 1 µg of a mixture of single and doublestranded [ ³H] E. coli DNA and a minimum of 50 units of AflIII incubated for 4 hours at 37ºC releases
Ligation and Recutting (Terminal Integrity) - After a 5-fold over-digestion of Lambda DNA with AflIII, >95% 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 AflIII.
Non-Specific DNase Activity (16 Hour) - A 50 µl reaction in NEBuffer 3.1 containing 1 µg of Lambda DNA and a minimum of 30 Units of AflIII incubated for 16 hours at 37ºC results in a DNA pattern free of detectable nuclease degradation as determined by agarose gel electrophoresis.