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Team:Paris Bettencourt/Notebook/Trojan Horse/Wednesday 31st July.html
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Latest revision as of 20:06, 3 October 2013
Trojan Horse
ASDF31th July
Vincent and Aude
Conjugation MGZ1, F+ & MG1655:
Protocol :
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From O/N cultures Dilute both strains 1/100 , in LB
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Wait for OD to reach O,2
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Prepare 4 tubes (in BD tubes) :
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Tube 1 = 0,5mL LB with Strain 1 (Here MGZ1) + O,5mL LB = control
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Tube 2 = 0,5mL LB with Strain 2 (here sT004) + 0,5mL LB = control
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Tube 3 and 4= 0,5mL LB with Strain 1 (Here MGZ1) + 0,5mL LB with Strain 2 (here sT004
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Incubate 2 hours at 37°C
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Plate 50ul for controls, 50uL, 20ul, 10ul for mixed tubes on LB antiobiotics (here tet and spec)
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Incubate overnight at 37°C
Vincent
Infectiveness characterization experiments
-the night before: prepare O/N of F+ cells, prepare O/N of phagemid producing cells (in this case : sT007 )
-centrifugate phagemid producing cells
-filter the supernatant 0.45um, stock it at 4°C (it contains the phages)
-dilute 200x MGZ1, F+ from O/N
-wait until OD600 = 0.7
-immediately mix MGZ1 and the supernatant in different proportions
here we planned to try 1/100 (vol supernatant/vol cells), 1/1000 and 1/10000
Unfortunately the supernatant was dropped on the bench.
