(Thermus species DT)
5’-A T G A A (N)11-3’
3’-T A C T T (N) 9 -5’
Purified from E.coli strain that carries the cloned tspDTRI gene from Thermus sp. DT.
Reaction Temperature: 70°C
Prototype: TspDTI
Inactivation Temperature (20 min): --
Reaction Buffer:
1x TspDTI Buffer: 10 mM Tris-HCl (pH 8.5 at 25°C), 10 mM MgCl2, 1 mM dithiothreitol +enhancers (1).
Avoid multiple cycles of freezing/thawing of the stock reaction buffer /no more than 3 times/. Thawing should be performed at temperatures not exceeding 10°C. Recommended procedure is to divide the provided reaction buffer into smaller portions and preserve them at –70°C for long-term. Temperature of –20°C should be used only for short-term storage.
Note 1: It is required to puryfy DNA before digestion. We recommend PCR / DNA Clean-Up Purification Kit or Agarose-Out DNA Purification Kit.
Note 2: It is not recommended to use more than 2 units per 30 μl reaction. It is suggested to perform digestion for over 1 hr.
Note 3: To avoid DNA shift during electrophoresis caused by strong protein-DNA interaction, it is recommended to terminate reaction by addition of reaction stop solution (containing denaturing reagent, i.e. 0.2% SDS) followed by 20 minutes heat inactivation in 89°C.
Unit Definition: One unit is the amount of enzyme required to digest 1 µg of pUC19 DNA to obtain stable digestion pattern in 1 hr in a total reaction volume of 30 µl.
Assay Conditions: 10 mM Tris-HCl (pH 8.5 at 25°C), 10 mM MgCl2, 1 mM dithiothreitol. Incubation is at 70°C for 1 h in a reaction volume of 30 µl.
Storage Buffer: 20 mM Tris-HCl, (pH 8.3 at 25°C), 25 mM (NH4)2SO4, 25 mM KCl, 0,5 mM EDTA, 0,5 mM dithiothreitol, 0.02% Triton™X-100, 0.02% Tween™20, 0.02% Igepal, 50% (v/v) glycerol.
Storage Conditions: Store at –20°C.
Quality Control:
Total Non-specific Endonuclease and Exonuclease: Incubation of 10 units of TspDTI with 1 µg of pUC19 DNA substrate at 70°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: 2.5, 5 and 10 units units of TspDTI and 0.11 µg (0.8 pmol of 3’-ends) of lambda/HindIII fragments (3’-labeled with T4 DNA Polymerase and [³H]dGTP and [³H]dCTP), incubated for 1 hr at 70°C resulted in 0.0-slope of %-end label released per units of enzyme. Reaction volume 10 µl.
5’-Exonuclease/5’-Phosphatase: Incubation of 2.5, 5 and 10 units of TspDTI with 0.02 µg (0.15 pmol of 5’-ends) of [5’-³²P] lambda/HaeIII fragments for 1 hr at 37°C resulted in 0.02-slope of %-end label released per unit of enzyme. Reaction volume 10 µl.
References:
1. Skowron, P.M., Majewski, J., �ylicz-Stachula, A., Rutkowska, S.M., Jaworowska, I. and Harasimowicz-S�owi�ska, R.I. (2003). Nucleic Acids Research 31, 14 e 74.