User:Sean P Corum/Notebook/PHIX174 Cell Free/2012/09/26: Difference between revisions

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(Autocreate 2012/09/26 Entry for User:Sean_P_Corum/Notebook/PHIX174_Cell_Free)
 
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==Entry title==
===Characterization B-C: Expression of PHIX174 promoters/UTRs fused to PX-UTR1-deGFP and PX-UTRX-deGFP.===
* Insert content here...


 
* Re-concentrated pBEST-Pr-MG-apt-UTR1-deGFP-T500. Quantifluore measured concentration to be 231.5 nM (1.6%).
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* Repeat cell-free expression, now with top plasmid concentration point actually at 10. Also, do it at gain range of 200, 225, and 250.
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* Cell-free expression 80%:
 
** 30 μL extract (12May11)
__NOTOC__
** 1.17 μL water
** 0.9 μL 100 mM Mg-glutamate (1 mM final)
** 2 μL 3 M K-glutamate (66.7 mM final)
** 22.5 μL 6 mM amino acids mix (1.5 mM final)
** 6.43 μL 14X 3-PGC buffer (1X final)
** 4.5 μL PEG8000 40% (2% final)
** 4.5 μL 200 μM malachite green (10 μM final)
* Aliquot 6 × 8 μL into 394-well plate and then add plasmid (pBEST-Pr-MGapt-UTR1-deGFP-T500) range:
*# 2 μL water = 0 nM
*# 2 μL 50 nM / 3^4 * 20% = .123 nM
*# 2 μL 50 nM / 3^3 * 20% = .370 nM
*# 2 μL 50 nM / 3^2 * 20% = 1.11 nM
*# 2 μL 50 nM / 3^1 * 20% = 3.33 nM
*# 2 μL 50 nM / 3^0 * 20% = 10 nM
* Plate reader:
*# 29 °C
*# Shake: fast double orbital 30s
*# Read: 625/655 nM @ gain = 200 w/ settings
*# Read: 625/655 nM @ gain = 225 w/ settings
*# Read: 625/655 nM @ gain = 250 w/ settings
*# Loop: 2-5 every 3 m for 12 h
** settings = full plate, high lamp energy, endpoint, bottom reading, other settings default

Revision as of 12:27, 26 September 2012

PHIX174 Cell Free Expression <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
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Characterization B-C: Expression of PHIX174 promoters/UTRs fused to PX-UTR1-deGFP and PX-UTRX-deGFP.

  • Re-concentrated pBEST-Pr-MG-apt-UTR1-deGFP-T500. Quantifluore measured concentration to be 231.5 nM (1.6%).
  • Repeat cell-free expression, now with top plasmid concentration point actually at 10. Also, do it at gain range of 200, 225, and 250.
  • Cell-free expression 80%:
    • 30 μL extract (12May11)
    • 1.17 μL water
    • 0.9 μL 100 mM Mg-glutamate (1 mM final)
    • 2 μL 3 M K-glutamate (66.7 mM final)
    • 22.5 μL 6 mM amino acids mix (1.5 mM final)
    • 6.43 μL 14X 3-PGC buffer (1X final)
    • 4.5 μL PEG8000 40% (2% final)
    • 4.5 μL 200 μM malachite green (10 μM final)
  • Aliquot 6 × 8 μL into 394-well plate and then add plasmid (pBEST-Pr-MGapt-UTR1-deGFP-T500) range:
    1. 2 μL water = 0 nM
    2. 2 μL 50 nM / 3^4 * 20% = .123 nM
    3. 2 μL 50 nM / 3^3 * 20% = .370 nM
    4. 2 μL 50 nM / 3^2 * 20% = 1.11 nM
    5. 2 μL 50 nM / 3^1 * 20% = 3.33 nM
    6. 2 μL 50 nM / 3^0 * 20% = 10 nM
  • Plate reader:
    1. 29 °C
    2. Shake: fast double orbital 30s
    3. Read: 625/655 nM @ gain = 200 w/ settings
    4. Read: 625/655 nM @ gain = 225 w/ settings
    5. Read: 625/655 nM @ gain = 250 w/ settings
    6. Loop: 2-5 every 3 m for 12 h
    • settings = full plate, high lamp energy, endpoint, bottom reading, other settings default
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