Imperial/Wet Lab/Protocols/CE1.5

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=Comparison of S30 home-made cell extract with the commercial S30 cell extract=
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==Aims==
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To test for the viability of the home made S30 cell extract and the amount of fluorescence it can express compared to the commercial S30 cell extract.
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==Equipments==
==Equipments==
-
===Equipments===
 
*Fluorometer + PC
*Fluorometer + PC
*1 Fluorometer plate (black)
*1 Fluorometer plate (black)
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*S30 cell extract (home made)
*S30 cell extract (home made)
*Reaction buffer (home made)
*Reaction buffer (home made)
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*Commercial S30 cell extract
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*Commercial S30 E.coli extract. Including:
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*Commercial Pre-incubation mix
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**175µl Amino Acid Mixture Minus Cysteine, 1mM
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*Amino Acids
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**175µl Amino Acid Mixture Minus Methionine, 1mM
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**Minus Cysteine
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**175µl Amino Acid Mixture Minus Leucine, 1mM
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**Minus Leucine
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**450µl S30 Extract, Circular (3 × 150µl)
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*pTet DNA plasmid
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**750µl S30 Premix Without Amino Acids
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*Nuclease Free water
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*DNA pTet-LuxR-pLux-GFP from midiprep
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*AHL stock solution 1mM
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==Steps==
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==Protocols==
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#Fill an eppendorf tube with two samples (56ul x 2) of home made cell extract including reaction buffer, pyruvate kinase and rNTPs.The volumes are shown below:
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#Fill an eppendorf tube with two samples (56ul x 2) of home made cell extract including reaction buffer, pyruvate kinase and rNTPs. The volumes are shown below:
#*Home made S30 - 16.2ul
#*Home made S30 - 16.2ul
#*Reaction Buffer- 30ul
#*Reaction Buffer- 30ul
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#*ddH<sub>2</sub> - 5.7ul
#*ddH<sub>2</sub> - 5.7ul
#Fill a second eppendorf tube with two samples (40ul x 2) of commercial cell extract including the amino acids mixture, and preincubation buffer.
#Fill a second eppendorf tube with two samples (40ul x 2) of commercial cell extract including the amino acids mixture, and preincubation buffer.
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#Place a quarter of each mixture in a well in the 96 well plate, making up 2 wells (B3 and B5).  
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===Loading Plate===
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#First read the background fluorescence of the 96-well plate using the fluorometer.
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#Choose suitable wells, with minimum fluorescence (30-40 au) to put the samples in. Do not use the wells at the edges and avoid putting samples in consecutive wells.
 +
#Follow the schematic for the plate and begin by loading the in vitro expression system into the correct wells. Before loading in the samples vortex the tubes for a few seconds to mix the solution.
 +
#Remove the lid from the 96-well plate and place in the fluorometer. Create a file name '''insert temp''' under:  D:\IGEM\'''INSERT DATE'''\Chassis testing . Export the data here. Each file should be named as the following:
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#* construct-temp-time-date
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#This measurement will give a background fluorescence measurement and can be used as our time zero data.
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#Place a quarter of each mixture in a well in the 96-well plate, making up 2 wells (B3 and B5).  
#In another well (C4), as a control, place another quarter of only the commercial cell extract, with 28ul of water.
#In another well (C4), as a control, place another quarter of only the commercial cell extract, with 28ul of water.
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#In the last well (D5), add a quarter of each mixture. This serves as the negative control.
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#In the second last well (D5), add a quarter of each mixture. This serves as the negative control.
#In the last well, add nuclease free water (again as a negative control).
#In the last well, add nuclease free water (again as a negative control).
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# In wells B3, B5 and C4, add 20ul of DNA.
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#In wells B3, B5 and C4, add 4ug worth of DNA.
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#Measure the fluorescence at 30 min intervals for the 1st hour, and hourly intervals thereafter.
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#In each of the wells add 3ul of AHL.
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#After each measurement, cover the plate with the sticky lid.
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#Top up all wells with nuclease free water to a total volume of 60ul.
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#Place the plate in the fluorometer to measure its initial fluorescence reading.  
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#After the measurement, place the sticky tape across the plate.
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#Before placing them in the water bath, wrap aluminium foil around them to prevent photobleaching.
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#Repeat the measurement every hour, for 6 hours.

Latest revision as of 02:15, 27 October 2007



Comparison of S30 home-made cell extract with the commercial S30 cell extract

Aims

To test for the viability of the home made S30 cell extract and the amount of fluorescence it can express compared to the commercial S30 cell extract.

Equipments

  • Fluorometer + PC
  • 1 Fluorometer plate (black)
  • Sticky seal tape
  • Gilson pipettes p200 p20 p10
  • Eppendorf Tubes
  • Stopwatch

Reagents

  • Pyruvate kinase
  • rNTPs
  • S30 cell extract (home made)
  • Reaction buffer (home made)
  • Commercial S30 E.coli extract. Including:
    • 175µl Amino Acid Mixture Minus Cysteine, 1mM
    • 175µl Amino Acid Mixture Minus Methionine, 1mM
    • 175µl Amino Acid Mixture Minus Leucine, 1mM
    • 450µl S30 Extract, Circular (3 × 150µl)
    • 750µl S30 Premix Without Amino Acids
  • Nuclease Free water
  • DNA pTet-LuxR-pLux-GFP from midiprep
  • AHL stock solution 1mM

Protocols

  1. Fill an eppendorf tube with two samples (56ul x 2) of home made cell extract including reaction buffer, pyruvate kinase and rNTPs. The volumes are shown below:
    • Home made S30 - 16.2ul
    • Reaction Buffer- 30ul
    • rNTP's - 1ul
    • Pyruvate Kinase - 3.1ul
    • DNA - 4ul
    • ddH2 - 5.7ul
  2. Fill a second eppendorf tube with two samples (40ul x 2) of commercial cell extract including the amino acids mixture, and preincubation buffer.

Loading Plate

  1. First read the background fluorescence of the 96-well plate using the fluorometer.
  2. Choose suitable wells, with minimum fluorescence (30-40 au) to put the samples in. Do not use the wells at the edges and avoid putting samples in consecutive wells.
  3. Follow the schematic for the plate and begin by loading the in vitro expression system into the correct wells. Before loading in the samples vortex the tubes for a few seconds to mix the solution.
  4. Remove the lid from the 96-well plate and place in the fluorometer. Create a file name insert temp under: D:\IGEM\INSERT DATE\Chassis testing . Export the data here. Each file should be named as the following:
    • construct-temp-time-date
  5. This measurement will give a background fluorescence measurement and can be used as our time zero data.
  6. Place a quarter of each mixture in a well in the 96-well plate, making up 2 wells (B3 and B5).
  7. In another well (C4), as a control, place another quarter of only the commercial cell extract, with 28ul of water.
  8. In the second last well (D5), add a quarter of each mixture. This serves as the negative control.
  9. In the last well, add nuclease free water (again as a negative control).
  10. In wells B3, B5 and C4, add 4ug worth of DNA.
  11. In each of the wells add 3ul of AHL.
  12. Top up all wells with nuclease free water to a total volume of 60ul.
  13. Place the plate in the fluorometer to measure its initial fluorescence reading.
  14. After the measurement, place the sticky tape across the plate.
  15. Before placing them in the water bath, wrap aluminium foil around them to prevent photobleaching.
  16. Repeat the measurement every hour, for 6 hours.
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