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Wet Lab: Protocols: Degradation Curve GFPmut3b

Aims:

To create a calibration curve to determine: [GFPmut3b] in vitro vs Fluorescence. This is essential for our data analysis to allow us to convert measurements we take in fluorescence, to molecules of GFPmut3b. To do this we need to add purified GFPmut3b into the in vitro chassis whilst maintainig a constant volume of 60ul. We can do this because we add 2ug of DNA in a 20ul solution

Equipment

• Fluorometer + PC
• 1 Fluorometer Plate (Black)
• 25°C water bath
• Sticky plate lid
• Eppendorf tubes
• Gilson p20,p200,p1000
• Stop watch

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
• GFPmut3b Recombinant Protein (1mg/ml)

Protocol

1. First collect all equipment and reagents and ensure that the fluorometer and that the PC connected has a data collection protocol installed.
2. For the cell extract, get the following out of the cell extract kit:
   • A.A's from kits
   • Premix tube
   • S30 tubes
3. For each reaction we will have 40ul Cell extract +17ul DNA (2ug DNA) + 3 ul GFPmut3b dilution.

Cell Extract

1. To prepare the commercial E.coli Cell Extract (for 10 reactions), carry out the following Procedure:
   
   • First prepare a complete amino acid mixture for the extract solution: Add the 25µl volume of two amino acid minus mixtures into an labeled eppendorf to give a volume of 50µl. Each amino acid minus mixture is missing one type of amino acid.
   • Add 200µl of S30 Premix (Without Amino Acid) into the eppendorf tube.
   • Then add 150µl of S30 Extract Circular too.
   • This mixture is for all the samples. Label the tube.
   • Any left over premix or cell extract should be returned to the freezer (biochemistry level 5) and labeled with new volumes.
   • Combine the Cell Extract and AA to give a final volume : 400µl

DNA Concentrations

1. Now Prepare the different DNA concentration, we are using an empty vector (330ng/µl) because we do not want any expression but do want to have DNA present to simulate the in vitro chassis
   • Remove 12.1µl of DNA in 170ul of total volume (make up with nuclease free water)
2. This can be added to cell extract to give a final volume of 570ul, this is enough to make 10 reactions of 57ul, each of which will be made upto 60ul when GFPmut3b dilution is added.

GFPmut3b Dilutions

1. Finally we need to prepare the GFPmut3b dilutions, we use a commercial recombinant protein of 1mg/ml. The final GFP concentrations we use are 1.85uM, 1.23uM, 0.62uM, 0.31uM and 0uM.

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. Add 57ul of cell extract+DNA solution to the correct wells following the schematic. When pipetting it is best to pipette straight down into the base of the wells and avoide getting solution on the sides of the wells.
4. Then add 3ul of the correct GFPmut3b dilution to the correct well following the schematic. Ensure that this is done as quickly as possible to avoid degradation.
5. Remove lid off the 96 well plate and place in the fluorometer. Create a file name GFP Degradation Curve under: D:\IGEM\INSERT DATE\Degradation\ 25oC. Export the data here.
6. Measure the plate in the fluorometer. This is the first reading.
7. Place the plate in the fluorometer to measure its initial fluorescent reading.
8. After the measurement, place the sticky tape across the plate, and put the plate in the 25oC water bath.
9. Before placing them in the water bath, wrap aluminium foil around them to prevent photobleaching.
10. Repeat the reading every 1 hour, until 6 hours have elapsed.