(Thermus aquaticus)
5’-G A C C G A (N)11-3’
3’-C T G G C T (N) 9 -5’
Purified from E.coli strain that carries the cloned taqRII gene from Thermus aquaticus*.
* patent pending
Reaction Temperature: 65°C
Prototype: TaqII
Inactivation Temperature (20 min): --
Reaction Buffer:
1x TaqII Buffer: 50 mM Tris-HCI (pH 8.5 at 25°C), 10 mM MgCl2, 1 mM dithiothreitol.
Note 1: It is required to puryfy DNA before digestion. We recommend PCR / DNA Clean-Up Purification Kitor Agarose-Out DNA Purification Kit.
Note 2: over 1 hr digestion is higly recommended. Best results are obtained with 3 hr digestion.
Unit Definition: One unit is the amount of enzyme required to digest 1 µg of pBR322 DNA to obtain stable digestion pattern in 1 hr in a total reaction volume of 30 µl.
Assay Conditions: 50 mM Tris-HCl (pH 8.5 at 25°C), 10 mM MgCl2, 1 mM dithiothreitol and 1 µg of pBR322 DNA. Incubation is at 65°C for 1 hr in a reaction volume of 30 µl.
Storage Buffer: 20 mM Tris-HCl (pH 7.5 at 25°C), 0.1 mM EDTA, 200 mM NaCl, 1 mM dithiothreitol, 200 µg/ml bovine serum albumin, 0.02% Triton™X-100, 0.02% Tween™20, 50% (v/v) glycerol.
Storage Conditions: Store at –20°C.
Quality Control:
Total Non-specific Endonuclease and Exonuclease: Incubation of 10 units of TaqII with 1 µg of pBR322 DNA substrate at 65°C for 16 hrs (a 160-fold over-digestion) resulted in the same sharp characteristic banding pattern as the standard assay reaction, as determined by agarose gel electrophoresis.
3’-Exonuclease: 5, 10 and 20 units of TaqII and 0.11 µg (0.8 pmol of 3’-ends) of lambda/TaqI fragments (3’-labeled with T4 DNA Polymerase and [³H]dGTP and [³H]dCTP), incubated for 1 hr at 65°C resulted in 0.0-slope of %-end label released per unit of enzyme. Reaction volume 10 µl.
5’-Exonuclease/5’-Phosphatase: Incubation of 5, 10 and 20 units of TaqII with 0.02 µg (0.15 pmol of 5’-ends) of [5’-³²P] lambda/HaeIII fragments for 1 hr at 65°C resulted in 0.02-slope of %-end label released per unit of enzyme. Reaction volume 10 µl.