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: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> FCS Measurements</span> | ||
|style="background-color: #F2F2F2" align="center"| | |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]] }}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}} | ||
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===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) | |||
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Latest revision as of 22:43, 26 September 2017
FCS Measurements | Main project page Previous entry Next entry |
July 1, 2013FCS Sample Preparation
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
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