Recombinant DNA Bacterial Transformation Student Instructions Plasmid Digest

1. Recombinant DNA Bacterial Transformation Student Instructions Plasmid Digest

2. Recombinant Transformation Plasmid Digest Obtain four 1.5 ml reagent tubes labeled: - pAmp - pKan - 2XRB”(Restriction Buffer) - B/H” (BamHI and HindIII enzymes).

3. Recombinant Transformation Plasmid Digest 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. NOTE: The buffer is always added before the enzymes. Tube 2X RB B/H __________________________________________________________ pAmp 7.5 µl 2 µl pKan 7.5 µl 2 µl

4. Recombinant Transformation Plasmid Digest Place the reaction tubes in a 37oC water bath and incubate for 30-60 minutes.

5. Recombinant Transformation Plasmid 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.