TspGW I

Source

Purified from E coli strain that carries the cloned tspGWRI gene from Thermus species GW

Unit Definition

One unit is the amount of enzyme required to digest 1 µg of pBR322 DNA to obtain a stable digestion pattern in 1 hour in a total reaction volume of 50 µl

Reaction Temperature

70°C

Heat Inactivation

No

Assay Unit Substrate

pBR322 DNA

Reaction Buffer

10 mM Tris-HCl (pH 8.5 at 25°C)
1 mM dithiothreitol
10 mM MgCl₂ 
+Enhancers (1) 

Notes:

1. Avoid multiple cycles of freezing / thawing of the stock reaction buffer; No more than three [3] times.
2. Thawing should be preformed at temperatures not exceeding 10°C.
3. Recommended procedure is to divide the provided reaction buffer into smaller portions and preserve them at -70°C for long term storage.
4. Recommended short term storage of reaction buffer is at -20°C.

Storage Buffer

20 mM Tris-HCl (pH 8.3 at 25°C)
25mM (NH4) 2SO4
0.5 mM EDTA
25 mM KCl
0.5 mM DTT
0.02%(v/v) Triton X-100, 0.02% (v/v) Tween 20,0.02% (v/v) Igepal CA-630
50% (v/v) glycerol

Assay Conditions 

10 mM Tris-HCl (pH 8.5 at 25°C)
10 mM MgCl₂ 
1 mM dithiothreitol
+enhancers
1 µg pBR322 DNA 
Incubation is at 70°C for 1 hour in a total reaction volume of 50 µl

Notes

1. It is not recommended to use more than 2 units per 50 µl reaction.
2. Digestion should be performed for over two [2] hours.
3. As TspGW I binds DNA very tightly, excess amount of TspGW I added can retard DNA migration on a gel, even in the presence of denaturing agents.
4. TspGW I 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.

Storage Conditions

Store at -20°C
Shipped on dry ice

References 

(1) Zylicz-Stachula, A., Harasimowicz-Slowinska, R., Sobolewski, I. and Skowron, P.M. (2002) Nucleic Acids Research 30, 7 e 33

Downloads

Certificate of Analysis PDF - Current Lot

Categories

Restriction Endonucleases