User:Madeleine Y. Bee/Notebook/Single Molecule Fluorescence/2013/07/01

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(FCS Data: Calibration Curves)
 
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> FCS Measurements</span>
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|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
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==July 1, 2013==
==July 1, 2013==
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===FCS Sample Preparation===
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*Green fluorescent beads
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**1000x diluted: from 10x dilution
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**10,000x diluted: from 10x dilution
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*Oligo D: 20,000pM → 1000pM → 150pM
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**150pM: from 1000pM dilution
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**125pM: from 150pM dilution
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**100pM: from 150pM dilution
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**80pM: from 150pM dilution
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**60pM: from 150pM dilution
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**50pM: from 150pM dilution
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**40pM: from 150pM dilution
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**30pM: from 150pM dilution
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**20pM: from 150pM dilution
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**10pM: from 150pM dilution
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*MB/DNA
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**1nM/800pM: from 2nM/1.6nM dilutions
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**900pM/720pM: from 50nM dilutions
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**800pM/640pM: from 50nM dilutions
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**700pM/560pM: from 2nM/1.6nM dilutions
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**600pM/480pM: from 2nM/1.6nM dilutions
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**500pM/400pM: from 2nM/1.6nM dilutions
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**400pM/320pM: from 2nM/1.6nM dilutions
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**300pM/240pM: from 2nM/1.6nM dilutions
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**200pM/160pM: from 2nM/1.6nM dilutions
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**100pM/80pM: from 2nM/1.6nM dilutions
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**75pM/60pM: from 2nM/1.6nM dilutions
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**50pM/40pM: from 2nM/1.6nM dilutions
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**25pM/12.5pM: from 2nM/1.6nM dilutions
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===FCS Data: Calibration Curves===
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[[Image:FCS_data_2013_0701_OD%2C_MB-DNA_calibrations.PNG]]<br.>
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Green fluorescent beads were run for 90s to align laser.<br.>
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Oligo D samples were run for 300s.<br.>
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MB-DNA samples were heated to ~70C for 25 min, cooled for 20 min, and run at 500s.<br.>
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====Notes====
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*Use green fluorescent beads to align laser
 +
*Calibration curves did not turn out as expected (linear trend)
 +
*DNA, not MB, should be in excess for DNA-MB samples to ensure all MB is bound
 +
*Focus on three concentrations, run each sample several times, average curves based on best spectra taken today
 +
**Oligo D: 150, 125, 100pM run three times at 500s per sample
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**DNA/MB: 100pM DNA/80pM MB, 75/60, 50/40pM run three times at 500s per sample (increase to 700s per sample if spectra are noisey)
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__NOTOC__

Current revision

FCS Measurements Main project page
Previous entry      Next entry

July 1, 2013

FCS Sample Preparation

  • Green fluorescent beads
    • 1000x diluted: from 10x dilution
    • 10,000x diluted: from 10x dilution
  • Oligo D: 20,000pM → 1000pM → 150pM
    • 150pM: from 1000pM dilution
    • 125pM: from 150pM dilution
    • 100pM: from 150pM dilution
    • 80pM: from 150pM dilution
    • 60pM: from 150pM dilution
    • 50pM: from 150pM dilution
    • 40pM: from 150pM dilution
    • 30pM: from 150pM dilution
    • 20pM: from 150pM dilution
    • 10pM: from 150pM dilution
  • MB/DNA
    • 1nM/800pM: from 2nM/1.6nM dilutions
    • 900pM/720pM: from 50nM dilutions
    • 800pM/640pM: from 50nM dilutions
    • 700pM/560pM: from 2nM/1.6nM dilutions
    • 600pM/480pM: from 2nM/1.6nM dilutions
    • 500pM/400pM: from 2nM/1.6nM dilutions
    • 400pM/320pM: from 2nM/1.6nM dilutions
    • 300pM/240pM: from 2nM/1.6nM dilutions
    • 200pM/160pM: from 2nM/1.6nM dilutions
    • 100pM/80pM: from 2nM/1.6nM dilutions
    • 75pM/60pM: from 2nM/1.6nM dilutions
    • 50pM/40pM: from 2nM/1.6nM dilutions
    • 25pM/12.5pM: from 2nM/1.6nM dilutions

FCS Data: Calibration Curves

Image:FCS_data_2013_0701_OD,_MB-DNA_calibrations.PNG
Green fluorescent beads were run for 90s to align laser.
Oligo D samples were run for 300s.
MB-DNA samples were heated to ~70C for 25 min, cooled for 20 min, and run at 500s.

Notes

  • Use green fluorescent beads to align laser
  • Calibration curves did not turn out as expected (linear trend)
  • DNA, not MB, should be in excess for DNA-MB samples to ensure all MB is bound
  • Focus on three concentrations, run each sample several times, average curves based on best spectra taken today
    • Oligo D: 150, 125, 100pM run three times at 500s per sample
    • DNA/MB: 100pM DNA/80pM MB, 75/60, 50/40pM run three times at 500s per sample (increase to 700s per sample if spectra are noisey)


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