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DpnI
(Diplococcus pneumoniae)
5'-G A T C- 3'
3'-C T A G- 5'
Purified from E.coli strain that carries the DpnI gene from Diplococus pneumoniae.
Reaction Temperature: 37°C
Prototype: DpnI
Inactivation Temperature (20 min): 80°C
Reaction Buffer:
1x Acet Buffer: 20 mM Tris-acetate (pH 7.5 at 37°C), 10 mM magnesium acetate, 50 mM potassium acetate, 1 mM dithiothreitol.
Unit Definition: One unit is the amount of enzyme required to completely digest 1 µg of pBR322 dam methylated DNA in 1 hr. Total reaction volume is 30 µl.
Notes:
- DpnI cleaves only methylated GATC sites;
- it may be necessary to add more enzyme to obtain complete digestion when using other buffer than optimal (Acet).
- it is recommended to add 10 u of the enzyme to obtain complete digestion of the methylated template after standard site-directed mutagenesis (total reaction volume is 50 µl)
Assay Conditions: 20 mM Tris-acetate (pH 7.5 at 37°C), 10 mM magnesium acetate, 50 mM potassium acetate, 1 mM dithiothreitol and 1 µg of pBR322 dam methylated DNA. Incubation is at 37°C for 1 hr in a reaction volume of 30 µl.
Storage Buffer: 10 mM Tris-HCl (pH 7.5 at 25°C), 1 mM dithiothreitol, 400 mM KCl, 0.1 mM EDTA, 0.1% Triton X-100, 200 µg/ml bovine serum albumin and 50%(v/v) glycerol.
Storage Conditions: Store at –20°C.
Quality Control:
All preparations are assayed for contaminating endonuclease, 3'-exonuclease, and nonspecific single- and double-stranded DNase activities.