User:Madeleine Y. Bee/Notebook/Single Molecule Fluorescence/2013/07/01: Difference between revisions
From OpenWetWare
(13 intermediate revisions by the same user not shown) | |||
Line 1: | Line 1: | ||
{|{{table}} width="800" | {|{{table}} width="800" | ||
|- | |- | ||
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> | |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> </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"|<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> </html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}} | ||
|- | |- | ||
| colspan="2"| | | colspan="2"| | ||
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> | <!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> | ||
== | ==July 1, 2013== | ||
=== | ===FCS Sample Preparation=== | ||
from | *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. ##### --> | <!-- ##### DO NOT edit below this line unless you know what you are doing. ##### --> |
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> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> |
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
|