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Team:Paris Bettencourt/Notebook/Trojan Horse/Thursday 18th July.html
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Latest revision as of 20:06, 3 October 2013
Trojan Horse
ASDF18th July
Vincent and Aude
Glycerol stock of ELS-41 and ELS-13 and MGZ1 now renamed sT004, sT005, sT006 respectively:
sT004: RP437, F+ Litmus28i_J23115-B0032-GFP AmpR
sT005: XL1-Blue, M13K07 KanR
Making electrocompetent cells from strain sT004, sT005 and MGZ1 => Bad old water => could not obtain competent cells.
Conjugation :
sT004: RP437, F+ Litmus28i_J23115-B0032-GFP + conjugate with MGZ1.
F+ comes from XL1 Blue (Ortiz paper) => F+ is tetR
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 100ul for controls, 100uL, 50ul, 20ul for mixed tubes on LB antiobiotics (here tet and spec)
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Incubate overnight at 37°C
Aude
Miniprep pT005 (Litmus28i_J23115-B0032-GFP) pT006 (M13K07) (Thermo Scientific Miniprep Kit)
pT005 : 280 ng /uL
pT006 : 61 ng/ul
Aude Vincent Sebastian and Yonatan
Work on the model
Define the players, the processes in place (more in Modeling file)
Anne and Clovis
Miniprep (sT002, sT003, NEB+pUC18) using the Thermo Scientific Miniprep Kit
1) Pellet 5ml of liquid culture (max speed, 1 min)
2) Discard supernatant
3) resuspend the cells in 250ul of resuspension olution
4) add 250ul of lysis solution, mix by inverting 4-6 times
5) add 350ul of neutralization solution
4) centrifuge for 5 min
5) transfer supernatant to spin column
6) centrifuge for 1 min
7) discard flow through
8) add 500 ul wash solution and centrifuge for 1 min , discard flow through(repeat this step)
9) centrifuge for 1 min to remove left over liquid
10) transfer the column on a 1.5ml tube
11) add 50ul of elution buffer and incubate for 2 min
12) centrifuge for 2 min
13) Nanodrop the concentration and freeze at -20°
pT.002: 53.9ng/ul -> redo with more liquid culture, it is only a low copy plasmid
pT.003: 53.8ng/ul -> redo with more liquid culture, it is only a low copy plasmid
pT.007: 214ng/ul
