User:Peixuan Pey/Sandbox

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Contents

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Peixuan Pey 11:23, 12 July 2007 (EDT) [1]

  1. Fallone CA. . pmid:11111111. PubMed HubMed [Paper1]


Protocol - In vitro testing - Operating Temperature Range and Initial Testing pT7 phase

Status

  • All tests for opertating range were completeted and the intial pT7 test carried out

Aims

  • To determine the exponential phase of production of GFP at extreme points of temperatures we are considering.
    • 10oC and 50oC for pTet in vitro
    • 37oC for pT7 in vitro

Equipments

  • Fluorometer + PC
  • Fridge at 4oC
  • Water bath in cold room at 10oC
  • 37oC shaking incubator
  • 50oC water bath
  • 3 Fluorometer plates (black)
  • 6 Sealing plate mats
  • Gilson pipettes 200, 20, 10
  • Eppendorf Tubes
  • Plate Centrifuge
  • 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
  • Commercial S30 T7 extract. Including:
    • 175µl Amino Acid Mixture Minus Cysteine, 1mM
    • 175µl Amino Acid Mixture Minus Methionine, 1mM
    • 175µl Amino Acid Mixture Minus Leucine, 1mM
    • 450µl T7 S30 Extract, Circular (3 × 150µl)
    • 750µl S30 Premix Without Amino Acids
  • MiiA water x1ml
  • GFP solution (For this initial experiment does not need to be purified GFP, we just want to know we have the right filter and that our settings are adjusted to measuring GFP)

Steps

  1. First collect all equipment and reagents and ensure that the fluorometer and that the PC connected has a data collection protocol installed.
  • Place one of the 96 well plate in the 10oC water bath, another n the 37oC incubator, and the last one in the 45oC incubator. This is done so as to heat them up to the appropriate temperature before the experiments start.
  1. Commercial E.coli Cell Extract: Take out 3 tubes of each component of the cell extract prepared earlier.
  2. Commercial E.coli Cell Extract: Pipette 20µl of each type of amino acid mix- AA without leucine + AA without cysteine, into an eppendorf tube. This is called the complete amino acid mixture.
  3. Commercial E.coli Cell Extract:Take another eppendorf tube and add 5µl of the E.coli complete amino acid mixture. Then add 20µl of S30 Premix Without Amino Acid. Then add 15µl of S30 Extract Circular. Place the eppendorf tube in a rack on the bench. Repeat the step for a total of 8 tubes.
  4. Commercial E.coli T7 Cell Extract: Repeat the above steps for the T7 E.coli cell extract. This time, take 2 tubes of prepared mixture; as well as prepare 10µl of amino acid mix each, to form 20µl of complete amino acid mixture.
  5. Vortex the tubes to mix thoroughly and place the 4 tubes of E.coli commercial extract in each incubator for ten minutes.
  6. Place 60μl of midipreped DNA plasmid (pT7) into an eppendorf tube and place it in the 37oC incubator.
  7. Place another 60μl of midipreped DNA plasmid (pTet) into two eppendorf tubes each and place one of them in the 10oC water bath, and the other in the 45oC water bath.
  8. Place 3 96 well plates into their respective incubators.

Loading Plate

  1. 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.
  2. Place the lid on the 96well plate and tape up the edges of the lid. This should be put into the incubator at 37oC for 10 minutes to allow temperature to equilibrate.
  3. Remove from incubators and spin-down in centrifuge in plate centrifuge at 2000rpm for a few seconds. Spin down is the process of bringing down any solution on lid or side of well into the base of the well. Alternatively can tap the top of the lid to bring down any solution to bottom of the well.
  4. Remove lid off the 96 well plate and place in the fluorometer. Create a file name protocol 2-1 under: D:\IGEM\INSERT DATE\CBD\ OTR. Export the data here. If repeated measurements change the second number to suit repeat number, e.g. 2nd repeat protocol 2-2, 5th repeat protocol 2-5. Once the data collection is set up then initiate the measurements.
  5. This measurement will give a back ground fluorescence measurement and can be used as our time zero data.
  6. 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.
  7. Place lid back on and place back in the respective incubators.
  8. After 30 minutes of incubation measure the fluorescence by repeating procedure 3-4 above. This initial measurement of 30 minutes is to find out how fast GFP is being produced. After this initial measurement, the intervals should be reassessed and adjusted accordingly.
  9. Before each measurement be careful to remember to either spin down or tap down the solution and to remove the lid before placing in the fluorometer.
  10. Repeat the above steps until 6 hours after the DNA was first added.
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