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

From OpenWetWare
Jump to: navigation, search
(July 1, 2013)
(fix raw html notebook nav)
 
(5 intermediate revisions by one other user not shown)
Line 2: Line 2:
 
|-
 
|-
 
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> FCS Measurements</span>
 
|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>}}
+
|style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}}
 
|-
 
|-
 
| colspan="2"|
 
| colspan="2"|
Line 9: Line 9:
 
===FCS Sample Preparation===
 
===FCS Sample Preparation===
 
*Green fluorescent beads
 
*Green fluorescent beads
**1000x diluted
+
**1000x diluted: from 10x dilution
**10,000x diluted
+
**10,000x diluted: from 10x dilution
*Oligo D
+
*Oligo D: 20,000pM → 1000pM → 150pM
**150pM
+
**150pM: from 1000pM dilution
**125pM
+
**125pM: from 150pM dilution
**100pM
+
**100pM: from 150pM dilution
**80pM
+
**80pM: from 150pM dilution
**60pM
+
**60pM: from 150pM dilution
**50pM
+
**50pM: from 150pM dilution
**40pM
+
**40pM: from 150pM dilution
**30pM
+
**30pM: from 150pM dilution
**20pM
+
**20pM: from 150pM dilution
**10pM
+
**10pM: from 150pM dilution
 
*MB/DNA
 
*MB/DNA
**1nM/800pM
+
**1nM/800pM: from 2nM/1.6nM dilutions
**900pM/720pM
+
**900pM/720pM: from 50nM dilutions
**800pM/640pM
+
**800pM/640pM: from 50nM dilutions
**700pM/560pM
+
**700pM/560pM: from 2nM/1.6nM dilutions
**600pM/480pM
+
**600pM/480pM: from 2nM/1.6nM dilutions
**500pM/400pM
+
**500pM/400pM: from 2nM/1.6nM dilutions
**400pM/320pM
+
**400pM/320pM: from 2nM/1.6nM dilutions
**300pM/240pM
+
**300pM/240pM: from 2nM/1.6nM dilutions
**200pM/160pM
+
**200pM/160pM: from 2nM/1.6nM dilutions
**100pM/80pM
+
**100pM/80pM: from 2nM/1.6nM dilutions
**75pM/60pM
+
**75pM/60pM: from 2nM/1.6nM dilutions
**50pM/40pM
+
**50pM/40pM: from 2nM/1.6nM dilutions
**25pM/12.5pM
+
**25pM/12.5pM: from 2nM/1.6nM dilutions
===FCS Data===
+
===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)
  
 
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->
 
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->

Latest revision as of 21:43, 26 September 2017

Owwnotebook icon.png FCS Measurements Report.pngMain project page
Resultset previous.pngPrevious entry      Next entryResultset next.png

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

FCS data 2013 0701 OD, 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)