IGEM:Paris Bettencourt 2012/Notebooks/suicide group/day by day//2012/09/21: Difference between revisions

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==Entry title==
==pLac + Colicin E2 Immunity Characterization==
* Insert content here...
We want to characterize the immunity by varying the concentration of IPTG and the ColicinE2 cells.
 
===Protocol===
#Grow overnight culture pSG.008 (ZI cells) with antibiotic (Cm).
#Centrifuge and resuspend in LB.
#Prepare 5 tubes of 10ml LB and put 10uL of the cells into each tube.
#Incubate 37C until the OD reaches 0.3
#Put IPTG with different concentration in each tube (0.1mM, 0.05mM, 0.025mM, 0.00625mM and 0/no IPTG).
#Incubate again until the OD reaches 1.0
#Take each 0.5ml and mix with 0.5ml Colicin cells (saturated, saturated diluted 1000x, plain LB) in small 2ml eppendorf tubes.
#Incubate for 30 minutes.
#Dilute 10000x (100x then 100x) in MgSO4 e-2M.
#Plate 200uL 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 10000 colonies at the end.
 
===Results===
The table shows the number of colonies survive in each plate IPTG x Colicin. This time we had too many colonies (very difficult to count), and still did not show the effect of Colicin and IPTG. The next experiment we would add another control which is cell with Cm resistance (pSB3C5) but no immunity gene, and do more dilution (and more carefully).
 
[[Image:SG_exp210912.png]]




Revision as of 17:16, 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 0.3
  5. Put IPTG with different concentration in each tube (0.1mM, 0.05mM, 0.025mM, 0.00625mM 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 1000x, plain LB) in small 2ml eppendorf tubes.
  8. Incubate for 30 minutes.
  9. Dilute 10000x (100x then 100x) in MgSO4 e-2M.
  10. Plate 200uL 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 10000 colonies at the end.

Results

The table shows the number of colonies survive in each plate IPTG x Colicin. This time we had too many colonies (very difficult to count), and still did not show the effect of Colicin and IPTG. The next experiment we would add another control which is cell with Cm resistance (pSB3C5) but no immunity gene, and do more dilution (and more carefully).


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