1. Recombinant DNA Bacterial Transformation Student Instructions Ligation

2. Recombinant Transformation Ligation Obtain reagent tubes labeled: - pAmp (digested) - pKan (digested) - 10X LB/ATP (ligation buffer) - T4 Lig (ligase) - DW (distilled water) Label a fresh sterile 1.5 ml tube “Lig” for the ligation reaction.

3. Recombinant Transformation Ligation Place the tubes marked “pAmp” and “pKan” in a 65 o C water bath for 10 minutes.

4. Recombinant Transformation Ligation Use the matrix below as a checklist while adding reagents to “Lig” tube for the ligation reaction. Make sure to add the reactants in the order listed from left to right. Add all reagents directly into the solution. Use a fresh pipette tip for each reagent. Tube pAmp pKan DW 10X LB/ATP T4 Lig ________________________________________________________ “Lig” 3 µl 3 µl 11 µl 4 µl 2 µl

5. Recombinant Transformation Ligation Incubate the reaction at room temperature for 24 hours. IMPORTANT: Make sure you do not throw away the tube marked “Lig” when you dispose of your other reagent tubes.

6. Recombinant Transformation Ligation If necessary, the ligation can be stored at -20 o C after room temperature incubation. Thaw the reaction before proceeding to transformation. For the best results, the transformation should take place immediately after incubation (the next day). Timing the events in this lab is very important.