Team:Wageningen UR/Journal/week2

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week 2: 7 may - 11 may

Office work

This is our first full week to work on this amazing project. There are four of us who can work whole days (Robert, Jeroen, Kees and Mark) and two of us who can work in the afternoon (Thijs and Wouter), the others will start when the sixth period end of our academic year.


This week we searched for programs which can predict the tertary structure of the monomers of the VLPs, we found several of them, but phyre2 really stand out with the simple fill in sheat and the possibility to download the PDB files of your predicted protein. Besides searching for programs, we also worked on the protocols we are going to use and check if we've got the required equipment and materials. Thijs and Mark worked also on the logo for the team and the backbone for the wiki. Jeroen and Wouter spent a day in the cold room to prepare large amounts of electrocompetent cells.

[Meeting]

written by: Mark

Lab work

Growing cells

Monday:

  • Checking plates
  • All plates grown well accept for amp plates, due to drying effects.
  • Growing E. coli in liquid LB. 3 plates per strain were picked and grown in 25 ml LB. All 3 strains in shaker, 180 RPM, 30 degree celcius.
  • Grow them overnight


Tuesday:

  • Continue growth.
  • Culture mach1 and DH5X are plated 10x and 1000x diluted on LB plates. Dilutions are done with demi water. Plates were placed in 37 degree Celcius stove.
  • Grow them overnight


Wednesday:

  • Check plates
  1. 10x dilution was to low, overgrown.
  2. 1000x was nearly overrown, but still acceptable.
  • Picked 1 MACH1 and 1 DH5X colony and grown in 10 ml SOB medium, 37 degree Celcius, 180 RPM
  • Grow them overnight


Preparing competent cells

Thursday:

  • Competent cells were prepared from Mach1 and DH5-Alfa E. coli.
  • following electrocompetent-cells protocol, with the following diviations:
  1. Starting culture: 4x 500 ml SOB
  2. Centrifuge rotor: SLA 3000


Testing CCMV protocol

Friday:

  • Inoculated at 8:45
  • IPTG at 11:45
  • Centrifugation in greiner tubes
  • Pellet stored at -20 degree Celcius, 50 ml greiner tubes
  • Miniprep following the Gene Jet Fermentas protocol
  1. pet28
  2. standard 10 stock 1
  3. standard 10 stock 7


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written by: Mark

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