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Latest revision as of 03:25, 27 October 2007

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Wet Lab: Protocols: Refined Testing for AHL Detection

Aims

• To determine the response of the pTet-LuxR-pLux-GFP construct, in-vitro, for different concentrations of AHL.
• To determine the response time of the construct in-vivo, for different AHL concentrations.

Day 1

Equipment

• Eppendorf Tubes
• Gilson pipettes p1000, p200, p20, p10

Reagents

• AHL stock solution of 1mM
• Nuclease free water

Protocol

• Using stock solution of 1mM of AHL, carry out the following dilutions using nuclease free water:
  1. Add 2ul of 1mM AHL to 998ul of water in an eppendorf tube, making up a 1000µl stock of 2000nM AHL.
  2. Remove 500µl from the 2000nM solution and put into another eppendorf tube with 500µl of water. This will give a 1000µl solution of 1000nM AHL.
  3. Remove 400µl from the 1000nM AHL solution and put in 600µl of water. This makes a 1000µl solution of 400nM AHL.
  4. Remove 151.5µl from the 1000nM AHL and put into an eppendorf with 348.5µl of water. This gives 500µl of 300nM AHL.
  5. Remove 500µl of 400nM AHL and put into an eppendorf with 500µl water. This makes 1000µl of 200nM AHL.
  6. Remove 500µl from 200nM AHL and put into an eppendorf tube with 500µl of water. The solution made is 1000µl of 100nM AHL.

Day 2

Equipments

• Fluorometer + PC
• Water bath in cold room at 25°C
• 1 Fluorometer plate (black)
• Sticky seal tape
• Gilson pipettes p200 p20 p10
• Eppendorf Tubes
• Stopwatch

Reagents

• 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
• AHL solutions 2000, 1000, 400, 300, 200 and 100nM
• DNA pTet-LuxR-pLux-GFP from maxiprep

Protocols

1. First collect all equipment and reagents and ensure that the fluorometer and that the PC connected has a data collection protocol installed.
2. Place the 96-well plates together with their plate mates in their respective incubators so as to heat them up to the appropriate temperature before the experiments start.
3. For the next step of the go to the biochemistry level 5 and remove:
   • A.A's from kits
   • Premix tubes (140ul)
   • S30 tubes (150ul)
4. Prepare the following cell extract mixture for all 21 reactions (one extra reaction):
   • First add 420µl of the premix solution into a labelled eppendorf.
   • Then add 315µl of the S30 cell extract mixture to S30 Premix Without Amino Acid.
   • Then prepare a complete amino acid mixture: Add 52.5μl of two amino acid minus mixtures into the eppendorf with the premix and S30 solution. Each amino acid minus mixture is missing one type of amino acid, and so by combining two solutions we are complementing each solution for the missing amino acid. Place eppendorf in a rack on bench.
   • Vortex the tubes to mix thoroughly.
   • Any leftover premix or cell extract should be returned to the freezer in biochemistry level 5 and labeled with new volumes.
5. Prepare the following dilution to get DNA concentration of 4µg:
   • Put 174µl of pLux DNA into a labelled eppendorf.
   • Add 166µl of nuclease free water into the DNA.
   • This will give a total volume of 340&micr;l, which is enough for all 18 samples with DNA in them.

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. Don't 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 lid off the 96-well plate and place in the fluorometer. Create a file named insert temp under: D:\IGEM\INSERT DATE\ID\ 25oC. 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. Then to begin the reaction add 4ug of purified DNA sample to each well indicated on the schematic. Be careful not to add to wells that DO NOT NEED DNA.
7. Add the appropriate amount of AHL to each well, so that the total reaction volume in each well is 60ul.
8. Place the plate in the fluorometer to measure its initial fluorescence reading.
9. After the measurement, place the sticky tape across the plate, and put the plate in the 25oC water bath.
10. Before placing them in the water bath, wrap aluminium foil around them to prevent photobleaching.
11. Repeat the measurement every 30 minutes, for 6 hours.