Daniel:Notebook/PosSequencing/2017-4-27: Difference between revisions
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Calculations for dye binding: | Calculations for dye binding: | ||
50 × 10<sup>-3</sup> × 500pmol⁄mg = 25 pmol | 50 × 10<sup>-3</sup> mg beads × <sup>500pmol</sup>⁄<sub>mg</sub> = 25 pmol available template @ max | ||
Line 17: | Line 17: | ||
<li>Put suspension on magnet</li> | <li>Put suspension on magnet</li> | ||
<li>Resuspend beads in 25 uL 4X SSC buffer</li> | <li>Resuspend beads in 25 uL 4X SSC buffer</li> | ||
<li>Make the following dye hybridization buffer</li> | |||
<ol type="a"> | |||
<li>2 each uL 10 uM dye; dcprobe6-488, dcprobe6-Cy3, dcprobe6-Cy5</li> | |||
<li>15 uL formamide</li> | |||
<li>4 uL nfH<sub>2</sub>O</li></ol> | |||
<li>Add 25 uL fluorescent probe in 60% formamide</li> | <li>Add 25 uL fluorescent probe in 60% formamide</li> | ||
<li>Incubate at room temp in the dark for 30 min</li> | <li>Incubate at room temp in the dark for 30 min</li> | ||
<li>Wash three times in 2X SSC</li> | <li>Wash three times in 2X SSC</li> | ||
<li>Resuspend in 50 uL 2XSSC</li> | |||
</ol> | </ol> | ||
<li>Imaging</li> | <li>Imaging</li> |
Latest revision as of 16:52, 27 April 2017
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