User:Madeleine Y. Bee/Notebook/Single Molecule Fluorescence/2013/06/18

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(June 18, 2013)
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==June 18, 2013==
==June 18, 2013==
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===FCS===
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*Calibration:
 +
**Alexa 532 Antibodies at 500pM and 100pM
 +
**green fluorescent beads diluted 10000x
 +
*Samples:
 +
**1-8 from 06/13/13
 +
No fluorescence obvserved.
 +
===Sample Prep: Remake 727.5nM DNA/9.7nM AuNPs===
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*Making 100mM DTT in 4% TEA
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*Extraction procedure with ethyl acetate
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*Combine equal volumes of 19.4nM AuNPs with 1455nM DNA from extraction to make 727.5nM DNA/9.4nM AuNP solution
 +
*Centrifuge four times, collecting four supernatants,at 4000rpm for 5 min intervals then re-dispersing in 50mM HEPES buffer
 +
*Take absorbance spectra for concentrated sample and four supernatants
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===Sample Prep: Remake 727.5nM DNA and 14.835nM AuNP Solutions===
 +
*Take fluorescence spectra, exciting at 532nm for the Molecular Beacon data
 +
===Sample Prep: Additional DNA Concentrations for DNA/ThT Correlation Graph===
 +
*Make the following solutions (use PBS for dilutions):
 +
**800nM DNA/2.5μM ThT
 +
**1091.25nM DNA/2.5μM ThT
 +
**1200nM DNA/2.5μM ThT
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Revision as of 12:27, 18 June 2013

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June 18, 2013

FCS

  • Calibration:
    • Alexa 532 Antibodies at 500pM and 100pM
    • green fluorescent beads diluted 10000x
  • Samples:
    • 1-8 from 06/13/13

No fluorescence obvserved.

Sample Prep: Remake 727.5nM DNA/9.7nM AuNPs

  • Making 100mM DTT in 4% TEA
  • Extraction procedure with ethyl acetate
  • Combine equal volumes of 19.4nM AuNPs with 1455nM DNA from extraction to make 727.5nM DNA/9.4nM AuNP solution
  • Centrifuge four times, collecting four supernatants,at 4000rpm for 5 min intervals then re-dispersing in 50mM HEPES buffer
  • Take absorbance spectra for concentrated sample and four supernatants

Sample Prep: Remake 727.5nM DNA and 14.835nM AuNP Solutions

  • Take fluorescence spectra, exciting at 532nm for the Molecular Beacon data

Sample Prep: Additional DNA Concentrations for DNA/ThT Correlation Graph

  • Make the following solutions (use PBS for dilutions):
    • 800nM DNA/2.5μM ThT
    • 1091.25nM DNA/2.5μM ThT
    • 1200nM DNA/2.5μM ThT


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