Team:Paris Liliane Bettencourt/Notebook/2011/07/29/

From 2011.igem.org

Team IGEM Paris 2011

Contents

Cyrille

QCM

The transformation of the quick change has given a few clones on the plates where 200 muL was spread. This show again the poor quality of our competent cells. 10 of the most promising clones ( 8 from the tube 1 ) where taken and put in culture in 15 ml of LB+Amp.

The miniprep of the 10 clones where done in the afternoon. The concentration was between 300 and 600 ng/muL. Oleg helped me in the task.

MH1 competent cells preparation

20 MuL of MH1 competent cells taken from the Hervé le Hir laboratory where put in culture in 15 mL of LB medium for 3 hours. Two of the tubes will serve to make glycerols of the cells, whereas the two other serves as preculture for the competent production.

The protocol for the competent cells is the following:

  • Take 1mL of precultured cell in 200 mL. Let them growth for 2-3h until they reach the OD600 between 0,3 and 0,6.
  • Spin the culture 10 min at 4000 rpm
  • Resuspend the cells in 100mL of CaCl2 at 50 mM ( 5 mL CaCL2 1M qsp 100 mL, and filtered )
  • Leave 20 min in ice
  • Spin 10 min at 4000rpm at 4°C
  • Resuspend in 20mL of CaCl2-Glycerol-Eau ( 1mL CaCl2 1M + 3,8g glycerol qsp 20mL H2O and filter )
  • Aliquote 500 muL in a CO2 ice and ethanol

Preparation of 3 glycerols of MH1

3 tubes (one for the -80 and two for the -20°C) of MH1 where prepared for storage.

Transformation with MH1 cells of the K1430379 biobrick from the 2010 plate

As some MH1 where unfrozen, we used a bit of them to transform a sensitive biobrick we have very few. The protocol followed is the usual heat-chock transformation protocol. The LB medium was intoduced at 11:30 today. The product was plated 2h after. Evrything was putted in the 37 incubator.

Camille & Danyel

T7 amber

All transformations (including Axel's) showed no results whatsoever.
• Overnight PCR (QM) relaunched

tRNA

Results of miniprep: 

     ng/µL        ratio
    
    144,6         1.94
    220.1         1.91
    427.4         1.99
    352.9         1.99
    265.1         1.94
    287.6         1.91

The 6 minipreps were diluted and sent for sequencing.

Kevin

Miniprep of plasmids pSG20 - pFX234 - pDAG464 - pDAG479

Axel

transformation in subtilis

transformation with K090403: colonies (Cm=5µg/mL) transformation with pHM3 plasmid: biofilm (Tet=5µg/mL)



Retrieved from "http://2011.igem.org/Team:Paris_Liliane_Bettencourt/Notebook/2011/07/29/"