IGEM:Paris Bettencourt 2012/Notebooks/suicide group/day by day//2012/09/19: Difference between revisions

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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===
#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 0.2
#Put IPTG with different concentration in each tube (0.1mM, 0.05mM, 0.025mM, 0.0125mM 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 100x, saturated diluted 10000x) in small 2ml eppendorf tubes.
#Incubate for 30 minutes.
#Dilute 1e5x (1000x then 100x) in MgSO4 e-2M.
#Plate 20uL 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===
A bit weird and random.. INSERT TABLE HERE 




Revision as of 16:27, 22 September 2012

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Ligation

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 20uL 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

A bit weird and random.. INSERT TABLE HERE


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