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

From OpenWetWare
Jump to: navigation, search
(fix raw html notebook nav)
 
(7 intermediate revisions by one other user not shown)
Line 1: Line 1:
 +
{|{{table}} width="800"
 +
|-
 +
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> FCS Measurements</span>
 +
|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"|
 +
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
 
==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)
 +
 +
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->
 +
|}
 +
 +
__NOTOC__

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)