Lidstrom: Tecan Plate Reader

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(Fluorescence-based promoter tests)
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== About the Tecan Infinite f500 plate reader ==
== About the Tecan Infinite f500 plate reader ==
-
* This plate reader can do fluorescence tests.  
+
== Basics ==
-
* [http://www.tecan.com/platform/apps/product/index.asp?MenuID=1881&ID=2425&Menu=1&Item=21.2.10.2.1 Product literature], [http://www.tecan.com/platform/apps/product/index.asp?MenuID=1884&ID=2436&Menu=1&Item=21.2.10.2.5 machine specs].
+
* This plate reader can do absorbances, fluorescence tests, and kinetic tests.
-
* The plate reader software writes directly to an excel file you can save.  If you want to save the spreadsheet that comes out as a CSV, chose Windows .csv in excel.  The default .csv file has ^M as the newline delimeter, but Windows .csv has one line per row in the spreadsheet.
+
** It is capable of doing liquid handling, though nobody has from 2010-2015.  --[[User:Janet B. Matsen|JM]]
 +
* [https://biochem.wisc.edu/sites/default/files/facilities/bif/instrument_pdfs/Infinite_F500_Manual.pdf MANUAL], [http://www.tecan.com/platform/apps/product/index.asp?MenuID=1881&ID=2425&Menu=1&Item=21.2.10.2.1 Product literature], [http://www.tecan.com/platform/apps/product/index.asp?MenuID=1884&ID=2436&Menu=1&Item=21.2.10.2.5 machine specs].
 +
* The plate reader software writes directly to an excel file you can save.  If you want to save the spreadsheet that comes out as a CSV, chose "Windows .csv" in excel.  The default .csv file has ^M as the newline delimeter, but Windows .csv has one line per row in the spreadsheet.
 +
* Contact [https://plus.google.com/ Janet Matsen] if you are interested in R scripts to parse the data dumped out from the machine.
-
== BCA Assay for Protein concentration ==
+
== Applications ==
-
* Use the 560 nm excitation filter.
+
* promoter strengths
 +
* OD540, OD600
 +
* BCA Assay for Protein concentration
 +
** Use the 560 nm excitation filter.
 +
 
 +
== Tips ==
 +
* The software cannot tell what filters are in without user input.  '''It is crucial that users are diligent with keeping information about the filters updated both on the filter-holder (black rectangle stick) and in the computer.'''
 +
** The computer senses what excitation or emission filter is inserted, and has user-specified information stored about which wavelength and band-with each filter is. 
 +
** In 6/2015 the machine was giving errors when reading OD600.  It turned out the machine thought the 600nm filter was in position 3, but there was an opaque black plug in place of the filter.  Somebody had not updated the information in the computer.
 +
** Some positions in the excitation filter have no labels as of 6/2015.  There is no easy way to tell what these are now and those filters are unusable.  Do not lose track of any other information!!
 +
* Filter nomenclature goes something like 600 (20).
 +
** This indicates the mean wavelength is 600nm, and the band-width is 20nm.  So it lets 590 - 620nm through.
 +
* Do not ever use tape to label the filters.  You must use the special see-through pre-labled stickers that come with the equipment to label which filters are in which position.
 +
** In 6/2015 the machine stopped working.  After hundreds of dollars were spent on repair-person hours, it was discovered that some lab tape had fallen off a filter and covered a sensor.
== Fluorescence-based promoter tests ==
== Fluorescence-based promoter tests ==
Line 19: Line 35:
** You may have negative values for some samples.
** You may have negative values for some samples.
*** Is there a way to blank it??  
*** Is there a way to blank it??  
-
* Check your data:
+
* Check the linearity of your OD and fluorescence across cell dilutions:
-
** Check that OD scales linearly with the amount of cell suspension added
+
** Check that OD scales linearly with the amount of cell suspension added '''and has intercept = 0'''
-
** Check that fluorescence intensity scales linearly with the amount of cell suspension added
+
*** make sure you subtract the OD of the media without cells first. 
-
** Then you know that fluorescence/OD should scale linearly with uL of cells
+
** Check that fluorescence intensity scales linearly with the amount of cell suspension added '''and has intercept = 0'''
-
*** make sure you subtract the OD of the media without cells first
+
*** correction via controls may be necessary if the intercept's magnitude isn't negligibly small.
 +
** If both are linear with intercept = 0, then you know that fluorescence/OD ratio will be a contant-ish number that doesn't depend on the cell concentration.
 +
*** If you divide the line y<sub>1</sub>=slope<sub>1</sub>*x<sub>OD</sub> + b<sub>1</sub> by y<sub>2</sub>=m<sub>2</sub>x<sub>OD</sub>+b<sub>2</sub>, and both b<sub>1</sub> and b<sub>2</sub> are zero, then the x<sub>OD</sub>s cancel out and a constant number is left behind.  '''This is the number you want to report.'''
** Then you can calculate fluorescence and summarize your results.  
** Then you can calculate fluorescence and summarize your results.  
** Sample data:  
** Sample data:  
Line 41: Line 59:
=== Sample Protocols ===
=== Sample Protocols ===
# See "Tips for Fluorescence Microplate Assays" in Invitrogen's [http://probes.invitrogen.com/media/publications/108.pdf Fluorescence Microplate Assays, 7th edition]
# See "Tips for Fluorescence Microplate Assays" in Invitrogen's [http://probes.invitrogen.com/media/publications/108.pdf Fluorescence Microplate Assays, 7th edition]
-
# Janet's protocol for RFP:  use filters we have, even though they aren't ideal. Wavelengths for fluorescent proteins: [http://flowcyt.salk.edu/fluo.html here].  
+
# Janet's protocol for RFP:  use filters we have, even though they aren't ideal.   (Note: different RFPs have different excitation/emission peaks).
## excitation = 535(25) nm.  10 flashes (default setting).
## excitation = 535(25) nm.  10 flashes (default setting).
## emission = 612(10) nm.  10 flashes (default setting).
## emission = 612(10) nm.  10 flashes (default setting).
-
## OD absorbance = 540nm (600 is also great)  
+
## OD absorbance = 600 (540nm is also great)  
 +
## Current as of 6/2015 --[[User:Janet B. Matsen|JM]]
[[image:2013_12_02 sample fluorescence assay protocol.png|thumb|center|upright=2|2013_12_02 sample fluorescence assay protocol.]]
[[image:2013_12_02 sample fluorescence assay protocol.png|thumb|center|upright=2|2013_12_02 sample fluorescence assay protocol.]]
# I subsequently learned that the z-position can be calculated automatically, meaning it can measure the height of the fluid.
# I subsequently learned that the z-position can be calculated automatically, meaning it can measure the height of the fluid.
 +
 +
=== Examples of Promoter testing in the Literature ===
 +
* Add examples here for future reference.
 +
* '''Is log-scale plotting appropriate?'''
 +
** I think Aaron Puri reported some promoter data on log scale in a 2014 group meeting. --'''[[User:Janet B. Matsen|JM]]'''

Current revision

Back to Protocols

Contents

About the Tecan Infinite f500 plate reader

Basics

  • This plate reader can do absorbances, fluorescence tests, and kinetic tests.
    • It is capable of doing liquid handling, though nobody has from 2010-2015. --JM
  • MANUAL, Product literature, machine specs.
  • The plate reader software writes directly to an excel file you can save. If you want to save the spreadsheet that comes out as a CSV, chose "Windows .csv" in excel. The default .csv file has ^M as the newline delimeter, but Windows .csv has one line per row in the spreadsheet.
  • Contact Janet Matsen if you are interested in R scripts to parse the data dumped out from the machine.

Applications

  • promoter strengths
  • OD540, OD600
  • BCA Assay for Protein concentration.
    • Use the 560 nm excitation filter.

Tips

  • The software cannot tell what filters are in without user input. It is crucial that users are diligent with keeping information about the filters updated both on the filter-holder (black rectangle stick) and in the computer.
    • The computer senses what excitation or emission filter is inserted, and has user-specified information stored about which wavelength and band-with each filter is.
    • In 6/2015 the machine was giving errors when reading OD600. It turned out the machine thought the 600nm filter was in position 3, but there was an opaque black plug in place of the filter. Somebody had not updated the information in the computer.
    • Some positions in the excitation filter have no labels as of 6/2015. There is no easy way to tell what these are now and those filters are unusable. Do not lose track of any other information!!
  • Filter nomenclature goes something like 600 (20).
    • This indicates the mean wavelength is 600nm, and the band-width is 20nm. So it lets 590 - 620nm through.
  • Do not ever use tape to label the filters. You must use the special see-through pre-labled stickers that come with the equipment to label which filters are in which position.
    • In 6/2015 the machine stopped working. After hundreds of dollars were spent on repair-person hours, it was discovered that some lab tape had fallen off a filter and covered a sensor.

Fluorescence-based promoter tests

Basics

  • Use a plate with a flat bottom but black well walls. The black walls makes sure the samples don't interfere with one another. This isn't an issue when excitation isn't used (e.g. absorbance measurements for BCA assay or cell culture density measurement).
  • If you have promoters in front of a fluorescent protein and want to test the promoter strength. The answer you get will depend (nonlinearly!) on:
    • The OD of the sample matters
    • The gain you chose matters
  • Should you subtract out fluorescence of the no-promoter control or divide the test value by the control?
    • Evidence of both can be found in the literature
    • You may have negative values for some samples.
      • Is there a way to blank it??
  • Check the linearity of your OD and fluorescence across cell dilutions:
    • Check that OD scales linearly with the amount of cell suspension added and has intercept = 0
      • make sure you subtract the OD of the media without cells first.
    • Check that fluorescence intensity scales linearly with the amount of cell suspension added and has intercept = 0
      • correction via controls may be necessary if the intercept's magnitude isn't negligibly small.
    • If both are linear with intercept = 0, then you know that fluorescence/OD ratio will be a contant-ish number that doesn't depend on the cell concentration.
      • If you divide the line y1=slope1*xOD + b1 by y2=m2xOD+b2, and both b1 and b2 are zero, then the xODs cancel out and a constant number is left behind. This is the number you want to report.
    • Then you can calculate fluorescence and summarize your results.
    • Sample data:
      • Quality check data.
        Quality check data.
      • Results.
        Results.

You should definitely:

  • Compare the test to a control at the same OD.
  • Compare across samples at the same OD
    • If you are comparing different cell types, (e.g. E. coli compared to a small methylotroph) you can't totally compensate for effects of cell size, but using the same OD should help.
  • This will require doing calculations for dilutions. You cannot just assume that 5uL of each type of cells + x uL water/buffer will give you the same OD.
  • Use at least 50 uL (100 is better) so the 96-well plate has enough fluid
  • Use the correct plate. Use one with black-sided wells & plastic that is compatible at your wavelength.

You should probably

  • put cells in a non-fluorescent buffer.
    • LB is known to contain fluorescent molecules.

Sample Protocols

  1. See "Tips for Fluorescence Microplate Assays" in Invitrogen's Fluorescence Microplate Assays, 7th edition
  2. Janet's protocol for RFP: use filters we have, even though they aren't ideal. (Note: different RFPs have different excitation/emission peaks).
    1. excitation = 535(25) nm. 10 flashes (default setting).
    2. emission = 612(10) nm. 10 flashes (default setting).
    3. OD absorbance = 600 (540nm is also great)
    4. Current as of 6/2015 --JM
2013_12_02 sample fluorescence assay protocol.
2013_12_02 sample fluorescence assay protocol.
  1. I subsequently learned that the z-position can be calculated automatically, meaning it can measure the height of the fluid.

Examples of Promoter testing in the Literature

  • Add examples here for future reference.
  • Is log-scale plotting appropriate?
    • I think Aaron Puri reported some promoter data on log scale in a 2014 group meeting. --JM
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