"

From 2013.igem.org

14/06/2013 - Making media

1. Calculate amount of powder of LB agar or LB broth mix required in accordance with manufacturer's instructions

2. Add LB powder to distilled water (we used 10 g of LB-broth or LB agar to 400ml of water

3. Autoclave the flask ( Make sure you have the autoclave tape on with label)

4. Add any antibiotic if needed once the flask has sufficiently cooled (approx 50 degrees).

19/06/2013 - Preparation of Chemically competent Cells

1. Grow the specific strain (BL21(DE3))on the plate. Plates will be in the incubator.

2. Select a colony from the plate with inoculating loop

3. The colony is dispersed in 100 ml LB broth in a conical flask.

4. The culture is left overnight- vigorous shaking at 37°C

Taking the OD of the grown culture

Want an OD of 0.1 in a culture volume of 100ml

Calculation:

V₁ x 3.2 = 100 x 0.1

V₁= 3.1 ml

96.9 ml [LB broth] + 3.1 ml [culture] = 100 ml culture with OD of 0.1

5. Take original OD volume of the culture (at 600 nm). Use LB as a blank. N.B: You need an OD less than 1.5 to measure the density accurately

6. Diluted culture is transferred to sterile flask. This is placed in shaking incubator at 37°C. After an hour, the OD is measured 15 minutes until it reaches the range of 0.4- 0.6

7. Centrifuge at 6000 xg for 10 min or 5500 xg for 15 min in 2 x 50 ml tubes.

8. Remove the supernatant from the tubes. Combine both the pellets and suspend in ice cold 0.1M CaCl₂

9. Leave the tube on ice for 30 min.

10. Spin cells at 6000 xg for 10 min or 5500 xg for 15 min and remove supernatant

11. Resuspend the pellet in 4 ml ice cold 0.1 CaCl2

12. Put on ice in cold room overnight

13. Add 1 ml sterile 100% glycerol

14. Make 50 µl aliquot and store at -80°C (after freezing with liquid nitrogen)

20/06/2013 - Knock Out Part 1 (Transformation of Chemically Competent E.Coli) </p> <p>21/06/2013 - Continued

24/06/2013 - Growing Cells From 21/06/2013

25/06/2013 - Electrically Competent Cells For Electroporation (E.Coli BL21 DE3)

26/06/2013 - Electroporation of PKD46 (lambda red recombinase plasmid) in E.Coli BL21 DE3 from 25/06/2013

27/06/2013 - Transformation Results

28/06/2013 - Further selection of Transformed cells and verification

01/07/2013 - Continued

04/07/2013 - Induction of Lambda Red Recombinase & Electro-Competency of transformed cells

09/07/2013 - PCR of Chloramphenicol and Homologous regions

10/07/2013 - Agarose Gel Electrophoresis of PCR product from 09/07

11/07/2013 - Received FAS Module - Plating up

12/07/2013 - FAS plate results

15/07/2013 - Stock of FAS cells for freezer

16/17 /07/2013 - DH5-Alpha cells grown up and FAS plasmid extraction using Qiagen MiniPrep Kit & Gel Electrophoresis of Product to verify

24/17 - Knockout Take II PCR of Chloromphenecol (New primers)

25/7 - PCR Of products - Worked

29/7 - Creating FAS Module Media

31/7 - Electroporation of cells with FAS