Restriction Mapping of a Bacterial Plasmid (Danna and Nathans, 1971)
Recombinant DNA Bacterial Transformation Student Instructions Plasmid Digest
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Transcript of Recombinant DNA Bacterial Transformation Student Instructions Plasmid Digest
Recombinant DNA Bacterial Transformation
Student InstructionsPlasmid Digest
Recombinant TransformationPlasmid Digest
Obtain four 1.5 ml reagent tubes labeled: - pAmp - pKan - 2XRB”(Restriction Buffer) - B/H” (BamHI and HindIII
enzymes).
Use the matrix below as a checklist while adding reagents to the pAmp and pKan tube.
Use a fresh pipette tip for each reagent. Add all reagents directly to the solution.
Recombinant TransformationPlasmid Digest
NOTE: The buffer is always added before the enzymes.
Tube 2X RBB/H
__________________________________________________________ pAmp 7.5 µl
2 µl
pKan 7.5 µl2 µl
Recombinant TransformationPlasmid Digest
Place the reaction tubes in a 37oC water bath and incubate for 30-60 minutes.
Recombinant TransformationPlasmid Digest
After incubation, freeze the reaction at -20oC until you are ready to proceed to Ligation. You can store reactions at this temperature for several days.