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Latest revision as of 02:59, 27 October 2007

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Wet Lab: Protocols: Packaging experiments

Aims

• Determine an optimum packaging technique for the experiments, that minimises the evaporation of the reaction mixture.
• Determine a suitable sampling technique which disrupts the reaction to a very minimum.
• The techniques that will be tested are:
  1. Plate with multiple layer of packaging tape - minimise evaporation
  2. Plate in which samples are pipetted, in single layer tape- see effect of pipetting on reaction
  3. Samples in PCR tubes with heated lid in a PCR machine - minimise evaporation but pipetting done
  4. Control - samples in 96 well plates with single tape layer and no pipetting

Equipments

• Fluorometer + PC
• 37°C incubator
• A PCR machine at 37°C
• 4 Fluorometer plates (black)
• Gilson pipettes p200 p20 p10
• Eppendorf and PCR Tubes
• Clear sticky tape
• Foil

Reagents

• Commercial S30 E.coli extract. Including:
  • 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
• DNA pTet-GFP
• Nuclease Free water

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 each of the 96 well plates together with their plate mats 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 experiment go to the biochemistry level 5 and remove:
   • A.A's from kits
   • 2xPremix tubes (60ul each)
   • 2xS30 tubes (45ul each)
4. For Each packaging and sampling tachnique Carry out the following Procedure
5. Commercial E.coli Cell Extract: First prepare a complete amino acid mixture for both extract solutions: Add the 5μl volume of two amino acid minus mixtures into an labeled eppendorf to give a volume of 10μl. 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.
6. Commercial E.coli Cell Extract: Take an eppendorf tube and add 10µl of the E.coli complete amino acid mixture. Then add 40µl of S30 Premix Without Amino Acid. Then add 30µl of S30 Extract Circular. Place the eppendorf tube in a rack on the bench.
7. Vortex the tubes to mix thoroughly and place the tubes of E.coli commercial extract in 37°C incubator for ten minutes.
8. Place an appropriate amount of the DNA plasmid into an eppendorf tube and place them in their respective incubators as well.

Loading Plate

1. Begin by loading the in vitro expression system into the correct wells of a 96 well plate. Before loading in the samples vortex the tubes for a few seconds to mix the solution.
2. Tap down the top of the plate to bring down any solution to bottom of the well.
3. Then to begin the reaction add 20μl of purified DNA sample to each well indicated on the schematic. Be careful not to add to wells that DO NOT NEED DNA.
4. Place the sticky tape across the plate. Put the plate in the 37oC incubator.
5. Mark the first plate as Plate 1.
6. Repeat the steps from 1-4 for another plate. Mark this plate as Plate 2.
7. Load another plate following the same procedure from 1-4, but put about 5 layers of sticky tape on. Mark this plate with Plate 3.
8. Put an empty plate (will be used to take readings of the samples in the PCR tubes) into the 37°C incubator too. Mark this plate as Plate 4.
9. Before placing them in the water bath, wrap aluminium foil around them to prevent photobleaching.
10. Take two eppendorf tubes and first put in 40µl of the cell extract mixture into each.
11. Now add 20&mirco;l of the DNA into each tube and place them in the PCR machine.
12. Turn the PCR machine on and set to 37°C.
13. Stagger the start of all the plates by around 5 minutes.
14. After 2 hours since the beginning of the reaction measure the fluorescence of each plate.
15. Take the plates out of their respective incubators. Remove lid off the 96 well plate and place in the fluorometer. Create a file name insert temp under: D:\IGEM\INSERT DATE\ID. Export the data here. Each file should be named as the following:
    • construct-temp-time-date
16. For Plate 2 take a measurement after 2 hours. Then pipette the samples into different wells and take the measurement again.
17. For Plate 4 reading, pipette the reaction mixture from the PCR tubes into the plate and then put in the fluorometer. Pipette the reaction mixture back into the PCR and place back into the PCR machine.
18. Measure the fluorescence every 2 hours for each plate.