Lab July 24 2009

9:55 am - subcultured the overnight culture of DH5α at approximately 1/100 (1 mL culture into approximately 99-100 mL) and put both flasks into the shaker

12.50 pm - Prepared 10 mL of CM1 buffer and CM2 buffer: Preparation of Competent Cells Protocol

1.13 pm - culture at 0.517 OD, started procedure.

Altered Transformation Protocol

2.17 pm - Dilute pUC18 plasmid to 20 uL at 1/10: 2 uL plasmid in 18 uL TE.

Add 3 uL diluted plasmid to each of the three tubes of 200 uL competent cells (two new, one old), and mix gently.

Incubate on ice for 30 min.

2.47 pm - Heat shock the mixes at 42oC for exactly 2 min.

Transfer to ice for 1-2 min.

Add 400 uL of 37oC LB.

2.55 pm - Incubate for 1 hr 15 min at 37 oC in a shaking water bath.

4.10 pm - Plate 0.2 mL on Amp + LB plates.

Incubate plates at 37 oC approximately 18-24 hr.

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