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Team:Paris Bettencourt/Notebook/Trojan Horse/Thursday 22nd August.html
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Latest revision as of 20:23, 3 October 2013
Trojan Horse
ASDF22nd August
Results :
All the transformation worked
Launch cultures (to do experiment)
-
mGZ1 F+ -Cola
-
MGZ1 F+ litmus + Helper
Launch overnigh culture to do glycerols.
exploring the parameters in play in the phage infectiveness of our new phagemid construct
-diluting 100x O/N of MGZ1, F+
-mix with 1/10 vol of supernatant containing phagemids with the cells with the following combination of parameters:
OD600 : 0.3 0.6 0.9
Plate at different time (min) after first contact with the supernatant: 2, 30, 60
Silencing experiments
- Launch O/N culture of producing phages cells ; centrifuge ; take supernatant and filter it.
-diluting 100x O/N of MGZ1, F+ -pCola in LB-Kan
- At OD 0,7 : 4 tubes : Ctrl, P1, P2 ,P3. Put 1 mL of cells in each tube. Centrifuge. Take out supernatant, resuspend in 1mL of LB. Add in tubes respectively : nothing, 100uL phages of Miniprep 1, Miniprep 2, miniprep 3.
- Incubate 45 minutes at 37°C
- Serial Dilute every tube until 10-5. Plate for each tube 100ul of 10-5 dilutions on Kan and LB and 100 ul of 10-2 and 10-3 dilution on Kan.
