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Revision as of 10:58, 23 October 2007

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Wet Lab: Protocols: Calibration 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 + Connected PC Turn on before beginning
• 96 well plate x1 + Plate lid
• 1.5ml eppendorf tube
• eppendorf rack
• 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 (at concentration determined friday) because we do not want any expression but do want to have DNA present to simulate the in vitro chassis
   • Remove ....µg 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 DsRed express dilutions, we use a commcerial recombinant protein of 1mg/ml. We need enough for 2 reactions and some excess so we will prepare 9ul:

#*1.95uM = 9ul DsRed express into an eppendorf tube

1. • 1.30uM = 6ul DsRed Express + 3ul of Nuclease Free WATER
   • 0.65uM = 3ul DsRed Express + 3ul of Nuclease Free Water
   • 0.32uM = 1.5ul DsRed Express + 7.5ul of Nuclease Free Water
   • 0uM = 9ul Nuclease Free Water

Loading Plate

1. First of all 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.
2. Then Add 3ul of the correct DsRed express dilution to the correct well following the schemtic. Ensure that this is done as quickly as possible to avoide degradation in Cell Extract.