Transformation Protocol For MC1061/P3 (E. coli strain)

1. Thaw bugs on ice.
While bugs are thawing:
Put transformation tubes (Falcon # 2059) on ice to pre-chill.
Make up fresh 500mM 2-ME: 2µl 2-ME + 55.6µl mQ H2O.
Mix cells gently by hand when thawed, and return to ice.

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

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