Talk:Knight:Beta-galactosidase assay/96 well format

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Current revision (15:16, 13 November 2007) (view source)
(A<sub>420</sub> versus o-nitrophenol concentration)
 
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==A<sub>420</sub> versus [[ONP|o-nitrophenol]] concentration==
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This is an outline of various control experiments that I need to do. It is a work in progress and has not been done.
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#Make up a solution of 7 mL to control for background absorbance in &beta;-galactosidase assays
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#*800 &mu;L permeabilization solution
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#*6 mL substrate solution without ONPG
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#*200 &mu;L EZ rich media supplemented with kanamycin and AHL
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#Make up 1mL of 4 mg/mL ONP.
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#Add 350 &mu;L of 4mg/mL solution to the first well.
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#Move 175 &mu;L of previous well to next well.
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#Add 175 &mu;L of background solution to that well.
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#Repeat dilution series until the well's solution looks totally clear.
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#Add an addditional well of 175 &mu;L background solution.
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#Measure A<sub>420</sub> and A<sub>550</sub> of each well in the plate reader.
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#Plot A<sub>420</sub> versus ONP concentration.  This relationship should be linear.
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Concentration series:
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4mg/mL -> 2mg/mL -> 1mg/mL -> 0.5 mg/mL -> 0.25 mg/mL -> 0.125 mg/mL -> 62.5 &mu;g/mL -> 31.25 &mu;g/mL -> 15.625 &mu;g/mL
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-> 7.8125 &mu;g/mL -> 3.90625 &mu;g/mL -> 1.953125 &mu;g/mL -> 0.9765625 &mu;g/mL
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==A<sub>600</sub> versus cell density==
==A<sub>600</sub> versus cell density==
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#Add 175 &mu;L of EZ Rich Media to that well.
#Add 175 &mu;L of EZ Rich Media to that well.
#Repeat dilution series until the well's solution looks totally clear.
#Repeat dilution series until the well's solution looks totally clear.
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#Add an addditional well of 175 &mu;L EZ Rich Media.
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#Add an additional well of 175 &mu;L EZ Rich Media.
#Measure A<sub>600</sub> of each well in the plate reader.
#Measure A<sub>600</sub> of each well in the plate reader.
#Plot A<sub>420</sub> versus dilution factor.  This relationship should be linear.
#Plot A<sub>420</sub> versus dilution factor.  This relationship should be linear.
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==Effect of evaporation on absorbance readings==
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[[Image:EvaporationEffectonA600.png|400px|right|thumb|This experiment was designed to assess the impact of evaporation upon the absorbance reading at 600nm of a culture sample in the [[Endy:Victor3 plate reader|Victor3 plate reader]].  A master culture of both pSB4K5-P20060 in MG1655 and pSB4K5-P20060.E0433 in TOP10 was grown.  For each construct, 50&mu;L of culture was aliquoted into each well of 3 rows in a 96 well plate.  Two rows were left empty.  To each well, between 0 and 100 &mu;L of H<sub>2</sub>O was added to the sample and the absorbance at 600nm was measured.  This setup was intended to mimic the situation in which there is a sample with a constant amount of absorbing material (i.e. cells) in the well, and there is evaporation of water over time from the well thereby changing both the concentration of material and volume of liquid (path length) in the well.  Thus, the variability in absorbance due to different amounts of H<sub>2</sub>O added should reflect the variability in absorbance due to evaporation in experiments.  This graph also shows 3 replicates of each combination of 50 &mu;L culture + X &mu;L of H<sub>2</sub>O to provide an indication of the variability between identical samples.  ''Click to view the larger image.'']]
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[[Image:EvaporationEffectonA430.png|400px|right|thumb|This experiment was designed to assess the impact of evaporation upon the absorbance reading at 430nm of an [[ONP]] solution in the [[Endy:Victor3 plate reader|Victor3 plate reader]].  50&mu;L of 0.75mg/mL ONP was aliquoted into each well of 2 rows in a 96 well plate (except column 12).  To each well, between 0 and 100 &mu;L of H<sub>2</sub>O was added to the sample and the absorbance at 600nm was measured.  This setup was intended to mimic the situation in which there is a sample with a constant amount of absorbing material (i.e. ONP) in the well, and there is evaporation of water over time from the well thereby changing both the concentration of material and volume of liquid (path length) in the well.  Thus, the variability in absorbance due to different amounts of H<sub>2</sub>O added should reflect the variability in absorbance due to evaporation in experiments.  This graph also shows 2 replicates of each combination of 50 &mu;L culture + X &mu;L of H<sub>2</sub>O to provide an indication of the variability between identical samples.  ''Click to view the larger image.'']]
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===Effect of evaporation on A<sub>600</sub> readings===
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#Aliquot 50 &mu;L of culture into an entire row of wells.
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#*Do duplicates rows to assess measurement variability in duplicate samples.
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#Aliquot 100 &mu;L of a of culture into a second row of wells.
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#Add increasing volumes of water to each well (by 10 &mu;L increments).
 +
#Measure the A<sub>600</sub> of the plate.
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 +
===Effect of evaporation on A<sub>420</sub> readings===
 +
#Aliquot 50 &mu;L of a set concentration of [[ONP]] into an entire row of wells.
 +
#*Could add different concentrations of ONP to different rows.
 +
#Add increasing volumes of water to each well (by 10 &mu;L increments).
 +
#Measure the A<sub>420</sub> of the plate.
 +
 +
==&beta;-galactosidase activity versus number of cells==
 +
#Do parallel &beta;-galactosidase assays with a variable volumes of cells from the same grown culture.
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#*1 = 1 &mu;L of cells
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#*2 = 2 &mu;L of cells
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#*3 = 3 &mu;L of cells
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#*4 = 4 &mu;L of cells
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#*5 = 5 &mu;L of cells
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#*6 = 5 &mu;L of media
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==&beta;-galactosidase activity versus growth phase of culture==
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#Grow an overnight culture of several constructs
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#*A = P20060 in MG1655
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#*B = P20060 in MG1655+IPTG
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#*C = P20060+IPTG+AHL
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#*D = P20060.E0433+IPTG
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#*E = P20060.E0433+IPTG+AHL
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#*F = R2000.E0433+IPTG+AHL
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#*G =
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#*H = Media+IPTG+AHL
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#In the morning, dilute back samples into EZ Rich Media to an A<sub>600</sub> of 0.001 (via a Nanodrop reading) in tubes (5 mL of culture in a 14mL tubes per construct).
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#Let grow 1 hour.
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#Add IPTG and/or AHL to cultures as appropriate.
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#Prepare 8mL of permeabilization buffer.
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#Every hour
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##Take 175 &mu;L of each culture and put it in the appropriate row and column of a 96 well plate (A600 plate).
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##Measure the absorbance at 600nm in the plate reader of the newest column of the A600 plate.
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##Aliquot 80 &mu;L permeabilization buffer into each well of the corresponding column of a second 96 well plate (permeabilization plate).
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##Take 20 &mu;L of culture from the most recent column of the A600 plate and add it to the corresponding column of the permeabilization plate.
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##Parafilm that column of the permeabilization plate.
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#Once each time point has been taken, move 25 &mu;L of each well from the permeabilized culture to a new plate.
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#Add 150 &mu;L of substrate solution to each well.
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#Place plate in the plate reader to measure change in A<sub>430</sub> as a function of time.
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#Plot the &beta;-galactosidase activity in Miller Units as a function of the A<sub>600</sub> of the culture.
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==A<sub>420</sub> versus [[ONP|o-nitrophenol]] concentration==
 +
 +
#Make up a solution of 14 mL to control for background absorbance in &beta;-galactosidase assays
 +
#*1600 &mu;L permeabilization solution
 +
#*12 mL substrate solution without ONPG
 +
#*400 &mu;L EZ rich media supplemented with kanamycin and AHL
 +
#Make up 1mL of 1 mg/mL ONP.
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#Add 350 &mu;L of 1mg/mL solution to the first well.
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#Move 175 &mu;L of previous well to next well.
 +
#Add 175 &mu;L of background solution to that well.
 +
#Repeat dilution series until the well's solution looks totally clear.
 +
#Add an additional well of 175 &mu;L background solution.
 +
#Measure A<sub>420</sub> of each well in the plate reader.
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#Plot A<sub>420</sub> versus ONP concentration.  This relationship should be linear.
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*'''[[User:Reshma P. Shetty|Reshma]] 13:16, 13 November 2007 (CST)''': I seem to get different calibration curves every time I do this experiment presumably due to errors in measurement of the ONP (since I am measuring small amounts).

Current revision

This is an outline of various control experiments that I need to do. It is a work in progress and has not been done.

Contents

A600 versus cell density

  1. Grow an overnight culture to saturation in EZ Rich Media
  2. Pellet the cells
  3. Resuspend in 1/4 of the original volume with EZ Rich Media
  4. Add 350 μL of cell suspension to the first well
  5. Add 175 μL of previous well to next well.
  6. Add 175 μL of EZ Rich Media to that well.
  7. Repeat dilution series until the well's solution looks totally clear.
  8. Add an additional well of 175 μL EZ Rich Media.
  9. Measure A600 of each well in the plate reader.
  10. Plot A420 versus dilution factor. This relationship should be linear.

Effect of evaporation on absorbance readings

This experiment was designed to assess the impact of evaporation upon the absorbance reading at 600nm of a culture sample in the Victor3 plate reader.  A master culture of both pSB4K5-P20060 in MG1655 and pSB4K5-P20060.E0433 in TOP10 was grown.  For each construct, 50μL of culture was aliquoted into each well of 3 rows in a 96 well plate.  Two rows were left empty.  To each well, between 0 and 100 μL of H2O was added to the sample and the absorbance at 600nm was measured.  This setup was intended to mimic the situation in which there is a sample with a constant amount of absorbing material (i.e. cells) in the well, and there is evaporation of water over time from the well thereby changing both the concentration of material and volume of liquid (path length) in the well.  Thus, the variability in absorbance due to different amounts of H2O added should reflect the variability in absorbance due to evaporation in experiments.  This graph also shows 3 replicates of each combination of 50 μL culture + X μL of H2O to provide an indication of the variability between identical samples.  Click to view the larger image.
This experiment was designed to assess the impact of evaporation upon the absorbance reading at 600nm of a culture sample in the Victor3 plate reader. A master culture of both pSB4K5-P20060 in MG1655 and pSB4K5-P20060.E0433 in TOP10 was grown. For each construct, 50μL of culture was aliquoted into each well of 3 rows in a 96 well plate. Two rows were left empty. To each well, between 0 and 100 μL of H2O was added to the sample and the absorbance at 600nm was measured. This setup was intended to mimic the situation in which there is a sample with a constant amount of absorbing material (i.e. cells) in the well, and there is evaporation of water over time from the well thereby changing both the concentration of material and volume of liquid (path length) in the well. Thus, the variability in absorbance due to different amounts of H2O added should reflect the variability in absorbance due to evaporation in experiments. This graph also shows 3 replicates of each combination of 50 μL culture + X μL of H2O to provide an indication of the variability between identical samples. Click to view the larger image.
This experiment was designed to assess the impact of evaporation upon the absorbance reading at 430nm of an ONP solution in the Victor3 plate reader.  50μL of 0.75mg/mL ONP was aliquoted into each well of 2 rows in a 96 well plate (except column 12).  To each well, between 0 and 100 μL of H2O was added to the sample and the absorbance at 600nm was measured.  This setup was intended to mimic the situation in which there is a sample with a constant amount of absorbing material (i.e. ONP) in the well, and there is evaporation of water over time from the well thereby changing both the concentration of material and volume of liquid (path length) in the well.  Thus, the variability in absorbance due to different amounts of H2O added should reflect the variability in absorbance due to evaporation in experiments.  This graph also shows 2 replicates of each combination of 50 μL culture + X μL of H2O to provide an indication of the variability between identical samples.  Click to view the larger image.
This experiment was designed to assess the impact of evaporation upon the absorbance reading at 430nm of an ONP solution in the Victor3 plate reader. 50μL of 0.75mg/mL ONP was aliquoted into each well of 2 rows in a 96 well plate (except column 12). To each well, between 0 and 100 μL of H2O was added to the sample and the absorbance at 600nm was measured. This setup was intended to mimic the situation in which there is a sample with a constant amount of absorbing material (i.e. ONP) in the well, and there is evaporation of water over time from the well thereby changing both the concentration of material and volume of liquid (path length) in the well. Thus, the variability in absorbance due to different amounts of H2O added should reflect the variability in absorbance due to evaporation in experiments. This graph also shows 2 replicates of each combination of 50 μL culture + X μL of H2O to provide an indication of the variability between identical samples. Click to view the larger image.

Effect of evaporation on A600 readings

  1. Aliquot 50 μL of culture into an entire row of wells.
    • Do duplicates rows to assess measurement variability in duplicate samples.
  2. Aliquot 100 μL of a of culture into a second row of wells.
  3. Add increasing volumes of water to each well (by 10 μL increments).
  4. Measure the A600 of the plate.

Effect of evaporation on A420 readings

  1. Aliquot 50 μL of a set concentration of ONP into an entire row of wells.
    • Could add different concentrations of ONP to different rows.
  2. Add increasing volumes of water to each well (by 10 μL increments).
  3. Measure the A420 of the plate.

β-galactosidase activity versus number of cells

  1. Do parallel β-galactosidase assays with a variable volumes of cells from the same grown culture.
    • 1 = 1 μL of cells
    • 2 = 2 μL of cells
    • 3 = 3 μL of cells
    • 4 = 4 μL of cells
    • 5 = 5 μL of cells
    • 6 = 5 μL of media

β-galactosidase activity versus growth phase of culture

  1. Grow an overnight culture of several constructs
    • A = P20060 in MG1655
    • B = P20060 in MG1655+IPTG
    • C = P20060+IPTG+AHL
    • D = P20060.E0433+IPTG
    • E = P20060.E0433+IPTG+AHL
    • F = R2000.E0433+IPTG+AHL
    • G =
    • H = Media+IPTG+AHL
  2. In the morning, dilute back samples into EZ Rich Media to an A600 of 0.001 (via a Nanodrop reading) in tubes (5 mL of culture in a 14mL tubes per construct).
  3. Let grow 1 hour.
  4. Add IPTG and/or AHL to cultures as appropriate.
  5. Prepare 8mL of permeabilization buffer.
  6. Every hour
    1. Take 175 μL of each culture and put it in the appropriate row and column of a 96 well plate (A600 plate).
    2. Measure the absorbance at 600nm in the plate reader of the newest column of the A600 plate.
    3. Aliquot 80 μL permeabilization buffer into each well of the corresponding column of a second 96 well plate (permeabilization plate).
    4. Take 20 μL of culture from the most recent column of the A600 plate and add it to the corresponding column of the permeabilization plate.
    5. Parafilm that column of the permeabilization plate.
  7. Once each time point has been taken, move 25 μL of each well from the permeabilized culture to a new plate.
  8. Add 150 μL of substrate solution to each well.
  9. Place plate in the plate reader to measure change in A430 as a function of time.
  10. Plot the β-galactosidase activity in Miller Units as a function of the A600 of the culture.

A420 versus o-nitrophenol concentration

  1. Make up a solution of 14 mL to control for background absorbance in β-galactosidase assays
    • 1600 μL permeabilization solution
    • 12 mL substrate solution without ONPG
    • 400 μL EZ rich media supplemented with kanamycin and AHL
  2. Make up 1mL of 1 mg/mL ONP.
  3. Add 350 μL of 1mg/mL solution to the first well.
  4. Move 175 μL of previous well to next well.
  5. Add 175 μL of background solution to that well.
  6. Repeat dilution series until the well's solution looks totally clear.
  7. Add an additional well of 175 μL background solution.
  8. Measure A420 of each well in the plate reader.
  9. Plot A420 versus ONP concentration. This relationship should be linear.
  • Reshma 13:16, 13 November 2007 (CST): I seem to get different calibration curves every time I do this experiment presumably due to errors in measurement of the ONP (since I am measuring small amounts).
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