Imperial/Wet Lab/Protocols/Prot1.3

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__NOTOC__
__NOTOC__
= Wet Lab: Protocols: Calibration Curve GFPmut3b =
= Wet Lab: Protocols: Calibration Curve GFPmut3b =
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'''Aims:''' <br>
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==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
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To create a calibration curve to determine: [GFPmut3b] in vitro vs Fluorescence. This is essential for our data analysis as it will 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.
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====Equipment====
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==Equipment==
*Fluorometer + PC  
*Fluorometer + PC  
*1 Fluorometer Plate (Black)  
*1 Fluorometer Plate (Black)  
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*Stop watch
*Stop watch
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====Reagents====
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==Reagents==
*Commercial S30 E.coli extract. Including:  
*Commercial S30 E.coli extract. Including:  
**175µl Amino Acid Mixture Minus Cysteine, 1mM  
**175µl Amino Acid Mixture Minus Cysteine, 1mM  
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*GFPmut3b Recombinant Protein (1mg/ml)  
*GFPmut3b Recombinant Protein (1mg/ml)  
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====Protocol====
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==Protocol==
#First collect all equipment and reagents and ensure that the fluorometer and that the PC connected has a data collection protocol installed.
#First collect all equipment and reagents and ensure that the fluorometer and that the PC connected has a data collection protocol installed.
#For the cell extract, get the following out of the cell extract kit:
#For the cell extract, get the following out of the cell extract kit:
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#For each reaction we will have 40ul Cell extract +17ul DNA (2ug DNA) + 3 ul GFPmut3b dilution.
#For each reaction we will have 40ul Cell extract +17ul DNA (2ug DNA) + 3 ul GFPmut3b dilution.
'''Cell Extract'''
'''Cell Extract'''
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#To prepare the commercial E.coli Cell Extract (for 10 reactions), carry out the following Procedure:<br>
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#To prepare the commercial E.coli Cell Extract (for 10 reactions), carry out the following procedure:<br>
#*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.   
#*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.  
#*Add 200µl of S30 Premix (Without Amino Acid) into the eppendorf tube.  
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#*Combine the Cell Extract and AA to give a final volume : 400&micro;l
#*Combine the Cell Extract and AA to give a final volume : 400&micro;l
'''DNA Concentrations'''
'''DNA Concentrations'''
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#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
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#Now Prepare the different DNA concentrations, we are using an empty vector (at concentration to be determined on Friday) because we do not want any expression but do want to have DNA present to simulate the in vitro chassis.
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#*Remove <font color=red> ....&micro;g</font> of DNA in 170ul of total volume (make up with nuclease free water)
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#*Remove 12.1&micro;l of DNA and make up a solution of total volume 170ul (made up with nuclease free water).
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#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.
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#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 topped up to 60ul when GFPmut3b dilution is added.
'''GFPmut3b Dilutions'''
'''GFPmut3b Dilutions'''
<br>
<br>
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Finally we need to prepare the GFPmut3b dilutions, we use a commercial recombinant protein of 1mg/ml. We need enough for 2 reactions and some excess so we will prepare 9ul:
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#Finally we need to prepare the GFPmut3b dilutions, we use a stock solution of protein of 1mg/ml. We need enough for 2 reactions and some excess so we will prepare 9ul:  
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#*0.2067uM = 0.0375ul GFPmut3b + 8.9625ul of Nuclease Free Water
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#*1.95uM = 9ul GFPmut3b into an eppendorf tube <font colour = red> -- how do you get 1.65uM?<font/>
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#*0.412uM = 0.075ul GFPmut3b + 8.925ul of Nuclease Free Water
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#*1.30uM = 6ul GFPmut3b + 3ul of Nuclease Free WATER
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#*0.62uM = 0.1125ul GFPmut3b + 8.887ul of Nuclease Free Water
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#*0.65uM = 3ul GFPmut3b + 3ul of Nuclease Free Water
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#*0.823uM = 0.15ul GFPmut3b + 8.85ul of Nuclease Free Water
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#*0.32uM = 1.5ul GFPmut3b + 7.5ul of Nuclease Free Water
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#*1.03uM = 0.18ul GFPmut3b + 8.82ul of Nuclease Free Water
#*0uM = 9ul Nuclease Free Water
#*0uM = 9ul Nuclease Free Water
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Chagne above to these concentrations: 0.2067; 0.412; 0.617; 0.823; 1.03
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===Loading Plate===
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-
 
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'''Loading Plate'''
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#First read the background fluorescence of the 96-well plate using the fluorometer.
#First read the background fluorescence of the 96-well plate using the fluorometer.
-
#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.
+
#Choose suitable wells, with minimum fluorescence (30-40 au) to put the samples in. Do not use the wells at the edges and avoid putting samples in consecutive wells.
-
#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.
+
#Add 57ul of cell extract + DNA solution to the correct wells. When pipetting it is best to pipette straight down into the base of the wells and avoid getting solution on the sides of the wells.
-
#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.  
+
#Then add 3ul of the correct GFPmut3b dilution to the correct well. Ensure that this is done as quickly as possible to avoid degradation.  
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#Remove lid off the 96 well plate and place in the fluorometer. Create a file name '''GFP Calibration Curve''' under:  D:\IGEM\'''INSERT DATE'''\Calibration\ 25oC. Export the data here.  
+
#Remove the lid of the 96-well plate and place in the plate in the fluorometer. Create a file name '''GFP Calibration Curve''' under:  D:\IGEM\'''INSERT DATE'''\Calibration\ 25oC. Export the data here.  
#Measure the plate in the fluorometer.
#Measure the plate in the fluorometer.

Latest revision as of 02:12, 27 October 2007



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 as it will 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)
  • 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 concentrations, we are using an empty vector (at concentration to be determined on Friday) 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 and make up a solution of total volume 170ul (made 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 topped up to 60ul when GFPmut3b dilution is added.

GFPmut3b Dilutions

  1. Finally we need to prepare the GFPmut3b dilutions, we use a stock solution of protein of 1mg/ml. We need enough for 2 reactions and some excess so we will prepare 9ul:
    • 0.2067uM = 0.0375ul GFPmut3b + 8.9625ul of Nuclease Free Water
    • 0.412uM = 0.075ul GFPmut3b + 8.925ul of Nuclease Free Water
    • 0.62uM = 0.1125ul GFPmut3b + 8.887ul of Nuclease Free Water
    • 0.823uM = 0.15ul GFPmut3b + 8.85ul of Nuclease Free Water
    • 1.03uM = 0.18ul GFPmut3b + 8.82ul of Nuclease Free Water
    • 0uM = 9ul Nuclease Free Water

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. Do not 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. When pipetting it is best to pipette straight down into the base of the wells and avoid getting solution on the sides of the wells.
  4. Then add 3ul of the correct GFPmut3b dilution to the correct well. Ensure that this is done as quickly as possible to avoid degradation.
  5. Remove the lid of the 96-well plate and place in the plate in the fluorometer. Create a file name GFP Calibration Curve under: D:\IGEM\INSERT DATE\Calibration\ 25oC. Export the data here.
  6. Measure the plate in the fluorometer.
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