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

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(June 18, 2013)
Current revision (15:00, 20 June 2013) (view source)
 
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Replication Attempts</span>
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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

Absorbance and Fluorescence Data

  • Absorbance data taken for DNA/ThT samples, (needs to be rerun due to inconsistencies)and extracted AuNPs with DNA and four supernatants
    • Samples might be remade before re-running
  • Fluorescence data taken for DNA/ThT samples, DNA and ThT comparison


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