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
<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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