# IGEM:Paris Bettencourt 2012/Notebooks/suicide group/day by day//2012/09/19

(Difference between revisions)
 Revision as of 19:06, 24 September 2012 (view source) (→Results)← Previous diff Revision as of 19:15, 24 September 2012 (view source)Next diff → Line 29: Line 29: ===Results=== ===Results=== - The table shows the number of colonies survive in each plate IPTG x Colicin. We had a random result and a very few colonies except for the cells without IPTG, so we decided to repeat the experiment. + The table shows the number of colonies survive in each plate IPTG x Colicin. Again we had a slighty random result and a very few colonies (theoretically we should get ~200 colonies if all cells survive, and we expected the cells survive if we do not mix them with Colicin), so we decided to repeat the experiment with less dilution. - [[Image:SG_exp190912.png | 700px]] + [[Image:SG_exp200912.png | 700px]]

## Revision as of 19:15, 24 September 2012

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## pLac + Colicin E2 Immunity Characterization

We want to characterize the immunity by varying the concentration of IPTG and the ColicinE2 cells.

### Protocol

1. Grow overnight culture pSG.008 (ZI cells) with antibiotic (Cm).
2. Centrifuge and resuspend in LB.
3. Prepare 5 tubes of 10ml LB and put 10uL of the cells into each tube.
4. Incubate 37C until the OD reaches 0.2
5. Put IPTG with different concentration in each tube (0.1mM, 0.05mM, 0.025mM, 0.0125mM and 0/no IPTG).
6. Incubate again until the OD reaches 1.0
7. Take each 0.5ml and mix with 0.5ml Colicin cells (saturated, saturated diluted 100x, saturated diluted 10000x) in small 2ml eppendorf tubes.
8. Incubate for 30 minutes.
9. Dilute 1e5x (1000x then 100x) in MgSO4 e-2M.
10. Plate 40uL in Cm to kill the Colicin cells and leave the immune cells.

Note: 1ml of cells of OD 1.0 has 1e9 cells, so we expect if all cells survive we will get 200 colonies at the end.

### Results

The table shows the number of colonies survive in each plate IPTG x Colicin. Again we had a slighty random result and a very few colonies (theoretically we should get ~200 colonies if all cells survive, and we expected the cells survive if we do not mix them with Colicin), so we decided to repeat the experiment with less dilution.