Transformation of Chemically competent E. coli

Date Dec./4/2014
The fallowing protocol where used from invitrogen
One Shot® Mach1™-T1R Chemically Competent E. coli

Transforming Competent Cells

Perform the following before starting the transformation procedure:
• Equilibrate a water bath to 42°C.
• Warm the vial of S.O.C. Medium (supplied with the kit) to room temperature.
• Spread X-Gal onto LB agar plates containing antibiotic, if desired.
• Warm the selective plates in a 37°C incubator for 30 minutes (use one plate for each transformation). If you are including the pUC19 control, make sure that you have one LB agar plate containing 100 μg/ml ampicillin. Note: For optimal growth of Mach1™-T1R E. coli cells, it is essential that selective plates are prewarmed to 37°C prior to spreading.

Transformation Procedure

We recommend including the pUC19 control plasmid DNA supplied with the kit in your transformation experiment to verify the efficiency of the competent cells. Do not use these cells for electroporation.
1. Thaw, on ice, one vial of One Shot® Mach1™-T1R Chemically Competent E. coli for each transformation.

2. Add 1 to 5 μl of the DNA (10 pg to 100 ng) into a vial of One Shot® cells and mix gently. Do not mix by pipetting up and down. If you are transforming the pUC19 control, add 1 μl (10 pg) into a separate vial of One Shot® cells and mix gently.

3. Incubate the vial(s) on ice for 30 minutes.

4. Heat-shock the cells for 30 seconds at 42°C without shaking.

5. Remove the vial(s) from the 42°C bath and place them on ice for 2 minutes.

6. Add 250 μl of room temperature S.O.C. Medium to each vial.

7. Cap the vial(s) tightly and shake horizontally at 37°C for 1 hour at 225 rpm in a shaking incubator.

8. Spread 25-100 μl of the transformation mix on a prewarmed selective plate. Store the remaining transformation mix at +4°C. Additional cells may be plated out the next day, if desired.

•   E. coli strain DH 5 alfa with no plasmid and sensitive to Amp. inoculated on LB+Amp. used as a control.
• One Shot® Mach1™-T1R Chemically Competent E. coli was inoculated on LB agar only to test its viability.