Restriction Digest of DNA
Select restriction enzymes to digest your plasmid .
Determine an appropriate reaction buffer by reading the instructions for your enzyme .
In a 1.5mL tube combine the following : DNA ( all amounts are for a typical reaction ; your amount may vary depending on the enzymes ) .
Typical mixture for single digest : Nuclease - free Water , 16 ul ; 10X Buffer EcoRI2 ulDNA ( 0.5 / 1 ug / ul ) , 1 ul ; EcoRI0 . 5 - 2 ul ;
Typical Mixture for Double Digest : Nuclease - free Water , 15 ul ; 10X Buffer , 02 ul ; DNA ( 0.5 - 1 ug / ul ) , 1 ul ; EcoRI , 1 ul ; PstI , 1 ul ;
Mix gently by pipetting .
Incubate tube at appropriate temperature ( usually 37°C ) for 1 hour .
