Team:Wageningen UR/Journal/week4

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= week 4: 21 may - 27 may  =
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= week 4: 21 may - 25 may  =
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== office work ==
== office work ==
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<p align="justify">
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This week felt a bit of a slow week. Two of our team members were a day sick. After the great success with the phyre2 program, we searched for a program that uses the output of the phyre2 program to predict the quaternary structure of the VLPs. In other words can a VLP be formed if we apply certain . We found out that there aren't many programs that can accurately predict the quaternary structure and the programs that are available aren't really compatible with phyre2.<br>
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Besides getting stuck with the prediction model Mark is investigating further for a new methodology to determine if VLPs were formed. He found two methods that can be used: asymmetric flow field-flow fractionation (AFFFF) followed  by multi angle light scaterring (MALS) and dynamic light scatering (DLS).<br>
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Thijs and Jasper continued further with the development of The Constructor and they hope to have he program ready for bet testing after the weekend
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</p>
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[meeting]
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written by: Mark
== lab work ==
== lab work ==
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21 may
 
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Sonified 6 x 30 seconds, output 3-4, amplitude around 20 on ice! cool as much possible, 50 seconds on ice between 30 second steps.
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'''Testing CCMV protocol'''
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lysate in 50 ml disassembly buffer.
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Centrifuged at 15000 RPM, 3 minute
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Monday:
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25 ml is dialised, 1st time, overnight.
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*Sonification of cells grown last week
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* specifications: 6 x 30 seconds, output 3-4, amplitude around 20 on ice! cool as much possible, 50 seconds on ice between 30 second steps.
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*Lysate dissolved in 50 ml disassembly buffer.
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*Centrifuged at 15000 RPM, 3 minute
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*Start of dialysis
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Tuesday:
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22 may
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*Took a negative control for SDS, non induced BL21 ''E.coli'' with CCMV wt,
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*Grown overnight in liquid LB+Kanamycin
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Negative control for SDS
 
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Non induced BL21 CCMV
 
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grew overnight in LB+kan
 
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follow protocol CCMV from day 1, after induction
 
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up to the 4th step of day 2
 
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sample transferred to new tube
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*Sample transferred to new tube
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stored in iGEM -20 degree Celcius as
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*Stored in -20 degree Celcius
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BL21 negative - iGEM SDS
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24 may
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Thursday:
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BL21 Plated and greiner tube 10 ml tube
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*BL21 plated and grown in Greiner tube 10 ml tube
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<ol>
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<li>BL21 2 plates freezer</li>
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<li>BL21 2 Greiner tubes 30 degree Celcius stove</li>
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</ol>
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*SDS-PAGE to check for the abundance of CCMV wt capsid proteins
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**6 different samples
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**10% SDS gel for proteins of 10 to 100 kDa size
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**staining for 15 min with Coomassie blue following rinsing with MQ water and 15 min of destaining
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BL21 2 plates freezer
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----
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BL21 2 Greiner tubes 30 degree Celcius stove
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[[https://2012.igem.org/Team:Wageningen_UR/Journal/week3 previous week]]          [[https://2012.igem.org/Team:Wageningen_UR/Journal/week5 next week]]

Latest revision as of 22:40, 26 September 2012

week 4: 21 may - 27 may

office work

This week felt a bit of a slow week. Two of our team members were a day sick. After the great success with the phyre2 program, we searched for a program that uses the output of the phyre2 program to predict the quaternary structure of the VLPs. In other words can a VLP be formed if we apply certain . We found out that there aren't many programs that can accurately predict the quaternary structure and the programs that are available aren't really compatible with phyre2.
Besides getting stuck with the prediction model Mark is investigating further for a new methodology to determine if VLPs were formed. He found two methods that can be used: asymmetric flow field-flow fractionation (AFFFF) followed by multi angle light scaterring (MALS) and dynamic light scatering (DLS).
Thijs and Jasper continued further with the development of The Constructor and they hope to have he program ready for bet testing after the weekend

[meeting]

written by: Mark

lab work

Testing CCMV protocol

Monday:

  • Sonification of cells grown last week
  • specifications: 6 x 30 seconds, output 3-4, amplitude around 20 on ice! cool as much possible, 50 seconds on ice between 30 second steps.
  • Lysate dissolved in 50 ml disassembly buffer.
  • Centrifuged at 15000 RPM, 3 minute
  • Start of dialysis


Tuesday:

  • Took a negative control for SDS, non induced BL21 E.coli with CCMV wt,
  • Grown overnight in liquid LB+Kanamycin


  • Sample transferred to new tube
  • Stored in -20 degree Celcius


Thursday:

  • BL21 plated and grown in Greiner tube 10 ml tube
  1. BL21 2 plates freezer
  2. BL21 2 Greiner tubes 30 degree Celcius stove
  • SDS-PAGE to check for the abundance of CCMV wt capsid proteins
    • 6 different samples
    • 10% SDS gel for proteins of 10 to 100 kDa size
    • staining for 15 min with Coomassie blue following rinsing with MQ water and 15 min of destaining

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