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.