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From 2012.igem.org

week 2: 7 may - 13 may

Office work

This is our first full week to work on our 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 join when the sixth period of our academic year ends .

This week we searched for programs which are able to predict the tertiary structure of the VLP monomers. Several of them were very interesting, but Phyre2 really stands out with the simple fill-in sheet and the possibility to download the PDB files of the predicted protein. Besides searching for programs, we also worked on the protocols that are going to be used and check if the required equipment and materials are available. Thijs and Mark worked also on the logo for the team and the template for the team-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 have grown well accept for ampicillin-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 have been grown a in shaker overnight at 180 RPM and 30 degree celcius.

Tuesday:

• Continue growth.
• Cultures of MachI and DH5a are plated 10x and 1000x diluted on LB plates. Dilutions are done with MQ water. Plates were placed in 37 degree Celcius stove and grown overnight.

Wednesday:

• Check plates

1. 10x dilution was too low, overgrown.
2. 1000x was nearly overgrown, but still acceptable.

• Picked 1 MachI and 1 DH5a colony and grown in 10 ml SOB medium, 37 degree Celcius, 180 RPM, overnight

Preparing competent cells

Thursday:

• Competent cells were prepared from both E. coli MachI and DH5a.
• following electrocompetent-cells protocol, with the following deviations:

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

plasmid	concentration (ng/µl)	260/280
pET28 (CCMV wt)	24.4	1.86
standard 10 stock 1	131.4	1.88
standard 10 stock 2	80.3	1.87

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written by: Mark