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

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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> FCS Measurements</span>
|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==
===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%2C_MB-DNA_calibrations.PNG]]<br.>
Green fluorescent beads were run for 90s to align laser.<br.>
Oligo D samples were run for 300s.<br.>
MB-DNA samples were heated to ~70C for 25 min, cooled for 20 min, and run at 500s.<br.>
====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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__NOTOC__

Revision as of 13:23, 1 July 2013

FCS Measurements <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

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

<br.> Green fluorescent beads were run for 90s to align laser.<br.> Oligo D samples were run for 300s.<br.> MB-DNA samples were heated to ~70C for 25 min, cooled for 20 min, and run at 500s.<br.>

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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