(Thermus species GW)
5’-A C G G A (N)11-3’ 3’-T G C C T (N) 9 -5’
Purified from E.coli strain that carries the cloned tspGWRI gene from Thermus sp. GW.
Reaction Temperature: 70°C
Prototype: TspGWI
Inactivation Temperature (20 min): --
Reaction Buffer:
1x TspGWI Buffer: 10 mM Tris-HCl (pH 8.5 at 25°C), 1 mM dithiothreitol, 10 mM MgCl2 +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 Kitor Agarose-Out DNA Purification Kit.
Note 2: It is not recommended to use more than 2 units per 50 μl reaction. Digestion should be performed for over 2 hr. As TspGWI binds DNA very tightly, excess amount of TspGWI added can retard DNA migration on a gel, even in the presence of denaturing agents.
Note 3: Restriction endonuclease TspGWI is very highly stimulated by the presence of two restriction sites in opposite orientation. Both the distance between recognition sequences and their immediate neighborhood also affects the cleavage effectiveness. Single site substrates are cleaved slowly.
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 50 µl.
Assay Conditions: 10 mM Tris-HCl (pH 8.5 at 25°C), 10 mM MgCl2, 1 mM dithiothreitol and 1 µg of pBR322 DNA. Incubation is at 70°C for 1 hr in a reaction volume of 50 µl.
Storage Buffer: 10 mM Tris-HCl (pH 7.5 at 25°C), 0.1 mM EDTA, 50 mM NaCl, 1 mM dithiothreitol, 100 µg/ml bovine serum albumin and 50% (v/v) glycerol.
Storage Conditions: Store at –20°C.
Quality Control:
Total Non-specific Endonuclease and Exonuclease: Incubation of 2 units of TspGWI with 1 µg of pBR322 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 of TspGWI 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 1hr at 70°C resulted in 0.01-slope of %-end label released per unit of enzyme. Reaction volume 10 µl.
5’-Exonuclease/5’-Phosphatase: Incubation of 2,5, 5 and 10 units of TspGWI with 0.02 µg (0.15 pmol of 5’-ends) of [5’-³²P] lambda/HaeIII fragments for 1 hr at 70°C resulted in 0.02-slope of %-end label released per unit of enzyme. Reaction volume 10 µl.
References:
1. Żylicz-Stachula, A., Harasimowicz-S�owi�ska, R. Sobolewski, I. and Skowron, P., (2002). Nucleic Acids Research 30, 7 e 33.