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

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(A<sub>420</sub> versus o-nitrophenol concentration)
(Effect of evaporation on absorbance readings)
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==Effect of evaporation on absorbance readings==
==Effect of evaporation on absorbance readings==
[[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.'']]
[[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.'']]
===Effect of evaporation on A<sub>600</sub> readings===
===Effect of evaporation on A<sub>600</sub> readings===
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#Add increasing volumes of water to each well (by 10 &mu;L increments).
#Add increasing volumes of water to each well (by 10 &mu;L increments).
#Measure the A<sub>600</sub> of the plate.
#Measure the A<sub>600</sub> of the plate.
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===Effect of evaporation on A<sub>420</sub> readings===
===Effect of evaporation on A<sub>420</sub> readings===

Revision as of 19:55, 31 October 2007

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 = MG1655
    • B = 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 a 96 well plate. (Each well in a row should be identical with 175 μL of culture according to the list above.)
  3. Let grow 1 hour.
  4. Add IPTG and/or AHL to rows as appropriate.
  5. Prepare the permeabilization buffer and aliquot 80 &muL into each well of a 96 well plate.
    • Using a larger volume for permeabilization step because the samples are sitting for a while.
  6. Measure the A600 of the plate every 30-60 minutes or grow the plate in the plate reader and pause it every 30-60 mins.
    • Use a gas permeable plate cover. Leave this on for absorbance measurements?
  7. At each hour point, take 20 μL of the next column of culture and add it to the corresponding columns of the permeabilization plate.
  8. Once each time point has been taken, move 25 μL of each well from the permeabilized culture to a new plate.
  9. Add 150 μL of substrate solution to each well.
  10. Place plate in the plate reader to measure change in A420 as a function of time.
  11. Plot the β-galactosidase activity in Miller Units as a function of the A600 of the culture.

A420 versus o-nitrophenol concentration

This experiment was designed to calibrate the absorbance at 430nm versus the concentration of o-nitrophenol in the Victor3 plate reader.  A stock solution of 0.75mg/mL ONP was made as described (in the same solution used for β-galactosidase assays) and a two-fold serial dilution was made.  These A430 values have been background subtracted (using beta;-galactosidase assay solution only).  Click to view the larger image.
This experiment was designed to calibrate the absorbance at 430nm versus the concentration of o-nitrophenol in the Victor3 plate reader. A stock solution of 0.75mg/mL ONP was made as described (in the same solution used for β-galactosidase assays) and a two-fold serial dilution was made. These A430 values have been background subtracted (using beta;-galactosidase assay solution only). Click to view the larger image.
  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.
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