Team:Wageningen UR/Journal/week2

From 2012.igem.org

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{{Template:Ecoil}}
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{{Template:WUR}}
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= week 2: 7 may - 13 may =
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= week 2: 7 may - 11 may =
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== Office work ==
== Office work ==
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<p align="justify">
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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.
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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 .
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<br><br>
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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.
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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.
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[Meeting]
[Meeting]
written by: Mark
written by: Mark
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</p>
== Lab work ==
== Lab work ==
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*Checking plates
*Checking plates
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*All plates grown well accept for amp plates, due to drying effects.
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*All plates have grown well accept for ampicillin-plates, due to drying effects.
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*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.
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*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.
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*Grow them overnight
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*Continue growth.
*Continue growth.
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*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.
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*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.
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*Grow them overnight
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*Check plates
*Check plates
<ol>
<ol>
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<li>10x dilution was to low, overgrown.</li>
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<li>10x dilution was too low, overgrown.</li>
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<li>1000x was nearly overrown, but still acceptable.</li>
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<li>1000x was nearly overgrown, but still acceptable.</li>
</ol>
</ol>
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*Picked 1 MACH1 and 1 DH5X colony and grown in 10 ml SOB medium, 37 degree Celcius, 180 RPM
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*Picked 1 MachI and 1 DH5a colony and grown in 10 ml SOB medium, 37 degree Celcius, 180 RPM, overnight
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*Grow them overnight
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'''Making competent cells'''
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'''Preparing competent cells'''
Thursday:  
Thursday:  
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MACH1 competent cells
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*Competent cells were prepared from both ''E. coli'' MachI and DH5a.
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followin pinky's protocol. with the following diviations
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*following electrocompetent-cells protocol, with the following deviations:
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*4x 500 ml SOB
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<ol>
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*SLA 3000 rotor
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<li>Starting culture: 4x 500 ml SOB</li>
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<li>Centrifuge rotor: SLA 3000</li>
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OD harvest at: 0.5 -0.45
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</ol>
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Friday:
Friday:
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CCMV protocol
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*Inoculated at 8:45  
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inoculated at 8:45 IPTG at 11:45
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*IPTG at 11:45
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*Centrifugation in greiner tubes
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*Pellet stored at -20 degree Celcius, 50 ml greiner tubes
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*Miniprep following the Gene Jet Fermentas protocol
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centrifugation in greiner tubes
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{| class="wikitable" style="text-align: center"
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|- style="font-style: italic"
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|plasmid
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|concentration (ng/µl)
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| 260/280
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|-
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|pET28 (CCMV wt)
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|24.4
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|1.86
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|-
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|standard 10 stock 1
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|131.4
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|1.88
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|-
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|standard 10 stock 2
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|80.3
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|1.87
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|}
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pellet stored at -20 degree Celcius, 50 ml greiner tubes
 
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Miniprep following the Gene Jet Fermentas protocol
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----
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*pet28
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[[https://2012.igem.org/Team:Wageningen_UR/Journal/week1 previous week]]                          [[https://2012.igem.org/Team:Wageningen_UR/Journal/week3 next week]]
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*standard 10 stock 1
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*standard 10 stock 7
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written by: Mark
written by: Mark

Latest revision as of 22:36, 26 September 2012

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


[previous week] [next week]

written by: Mark

Retrieved from "http://2012.igem.org/Team:Wageningen_UR/Journal/week2"