Daniel:Notebook/PosSequencing/2017-4-27
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Emulsion PCR (Started Yesterday)[edit]
Protocol Part 3-Hybridization and Imaging[edit]
Probe hybridization and washes based on Matt's experiment.
Calculations for dye binding:
50 × 10-3 mg beads × 500pmol⁄mg = 25 pmol available template @ max
- Fluorescent Oligo Binding
- Put suspension on magnet
- Resuspend beads in 25 uL 4X SSC buffer
- Make the following dye hybridization buffer
- 2 each uL 10 uM dye; dcprobe6-488, dcprobe6-Cy3, dcprobe6-Cy5
- 15 uL formamide
- 4 uL nfH2O
- Add 25 uL fluorescent probe in 60% formamide
- Incubate at room temp in the dark for 30 min
- Wash three times in 2X SSC
- Resuspend in 50 uL 2XSSC
- Imaging