Talk:Team:IvyTech-South Bend/21 October 2010

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Preparation of Electrocompetent Cells

See Ausubel et al. (1987) and Miller and Nickoloff (1995) for additional information.

1. Inoculate 500 ml of L-broth with 1/100 volume of a fresh overnight E. coli culture.

2.Grow the cells at 37 °C shaking at 300 rpm to an OD60o of approximately 0.5-0.7 (the best results are obtained with cells that are harvested at early- to mid-log phase; the appropriate cell density therefore depends on the strain and growth conditions).

3.Chill cells on ice for-20 rain. For all subsequent steps, keep the cells as close to 0 °C as possible (in an ice/water bath) and chill all containers in ice before adding cells. To harvest, transfer the cells to a cold centrifuge bottle and spin at 4000 x g for 15 minutes at 4 °C.

4.Carefully pour offand discard the supematant. It is better to sacrifice the yield by pouring offa few cells than to leave any supematant behind.

5.Gently resuspend the pellet in 500 ml of ice-cold 10% glycerol. Centrifuge at 4000 x g for 15 minutes at 4 °C; carefully pour offand discard the supematant.

6.Resuspend the pellet in 250 ml of ice-cold 10% glycerol. Centrifuge at 4000 x g for 15~.minutes at 4 °C; careftiily pour offand discard the supernatant.

7. Resuspend the pellet in -20 ml of ice-cold 10% glycerol. Transfer to a 30 ml sterile Oakridge tube. Centrifuge at 4000 x g for 15 minutes at 4 °C; carefully pour off and discard the supernatant.

8. Resuspend the cell pellet in a final volume~t of ice-cold 10% glycerol. The cell concentration should be about 1-3 x 1010 cells/ml. This suspension may be frozen in aliquots on dry ice and stored at -70 °C. The cells are stable for at least 6 months under these conditions.

5.2 Electroporation

1. Thaw the cells on ice. For each sample to be electroporated, place a 1.5 ml microfuge tube and either a 0.1 or 0.2 cm electroporation cuvette on ice.

2.In a cold, 1.5 ml polypropylene microfuge tube, mix 40 pl of the cell suspension with 1 to 2 ~tl ofDNA (DNA should be in a low ionic strength buffer such as TE). Mix well and incubate on ice for -1 minute. (Note: it is best to mix the plasmids and cells in a microfuge tube since the narrow gap of the cuvettes prevents uniform mixing.)

3. Set the MicroPulser to "Ecl" whefi using the 0.1 cm cuvettes. Set it to "Ec2" or "Ec3" when using the 0.2 cm cuvettes. See Section 4 for operating instructions.

9/21/10

Pulling of LacZ plate 1 well 23F __ I732094 I added 10ul of di H2O to the well, did not pull 5 ul out because we did not have electorcom cells ready There is 10 ul of di H20 and lacZ in well Dgarvey