Team:Paris Bettencourt/Notebook/Trojan Horse/Tuesday 13th August.html

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Revision as of 12:34, 20 September 2013

Trojan Horse

13th August

Place your twit here

Clovis

Digestion of the PCR product (LacZalpha) with NcoI and BglII

PCR purification using Thermo Scintific Kit of the digestion → 6,2 ng/ul

Miniprep of pACYADuet-1 (pT002) using Thermo Scientific Kit → 132,7 ng/ul

Start O/N sT005 and sT010

Vincent

PCR of pUC18 (pT007) with iT0007 & iT0008 / PCR of litmus28i-GFP (pT005) with iT0005 & iT0006

PCR Gradient of 8 tubes +-5°C around 54°C for both PCRs (pUC18 and Litmus18i-GFP)

Failure, no bands appear on the gel.

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

  1. 1mL MGZ1,F+ + 100ul LB

  2. 1mL MGZ1,F+ + 100ul surnageant diluted 1/100

  3. 1mL MGZ1,F+ + 100ul surnageant diluted 1/1000

  4. 1mL MGZ1,F+ + 100ul surnageant diluted 1/1000

-incubate 45 minutes at 37°C for the protein to be expressed.

  • Serial dilute the tubes 10^-1, 10^-2, 10^-3, 10^-4, 10^-5, 10^-6

    1. 4 times 6 tubes with 900uL LB, add 100ul of one previously made tube, mix and transfert 100ul to next tube.

  • Plate on LB (10^-6), Kan( 10^-3), Amp(10^-5 - 10^-6), Kan and Amp(10^-2 - 10^-3)

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