The products of PCR reaction solution are further digested with
HinfI restriction enzyme.
1.A master mix for all enzyme reactions is prepared by adding
the volumes of the required solutions to one tube: 1.0mL 10×
buffer R, 0.5mLHinfI restriction enzyme (10 U/mL), and
3.5mL distilled water.
2.Add 5mL each PCR reaction solution into new labeled microcentrifuge tubes.
3.Aliquot 5mL of this master mix to each microcentrifuge tubes
(step 2). The reaction tube is centrifuged to bring solution to
the bottom of the tube.
4.Transfer all tubes to a water bath and incubated at 37 °C for
1 h and then chill the reaction tube on ice.