2. Aliquot 100µl cells into pre-chilled tube. Put excess bugs back into the -70 freezer.

3. Add 5µl of the 500mM 2-ME to the 100µl cells, swirl gently. Incubate on ice for 10 minutes, swirling every 2 minutes.

4. Add DNA (1 to 5 µl), swirl tube, incubate on ice for 30 minutes.

5. Heat shock the cells at 42°ree;C fo 75 seconds, then return tube to ice for 2 minutes. (Time varies for different quantities of cells- see Invitrogen protocol).

6. Add 900 µl SOC; Rescue at 37°ree;C for 1 hour, shaking.

7. Spread 100µl (& 20µl) on LB+Amp+Tet plates, --> 37°ree;C ON. Use Amp 50 µg/ml, Tet 15 µg/ml.

INFO: MC1061/P3 bugs are strain MC1061 that carry (stably) a P3 plasmid. P3 carries an amber AmpR and an amber TetR gene. The engineered plasmid with which you transform these bugs should carry a SupF gene, which allows a read-through of the amber codon, conferring Amp and Tet resistance to the transformed cells.

Shirley Reynolds