Daniel:Notebook/PosSequencing/2017-4-27: Difference between revisions

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Calculations for dye binding:  
Calculations for dye binding:  


50 &times; 10<sup>-3</sup> &times; 500pmol&frasl;mg = 25 pmol
50 &times; 10<sup>-3</sup> mg beads &times; <sup>500pmol</sup>&frasl;<sub>mg</sub> = 25 pmol available template @ max




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<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]

Back to Calendar

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 × 500pmolmg = 25 pmol available template @ max


  1. Fluorescent Oligo Binding
    1. Put suspension on magnet
    2. Resuspend beads in 25 uL 4X SSC buffer
    3. Make the following dye hybridization buffer
      1. 2 each uL 10 uM dye; dcprobe6-488, dcprobe6-Cy3, dcprobe6-Cy5
      2. 15 uL formamide
      3. 4 uL nfH2O
    4. Add 25 uL fluorescent probe in 60% formamide
    5. Incubate at room temp in the dark for 30 min
    6. Wash three times in 2X SSC
    7. Resuspend in 50 uL 2XSSC
  2. Imaging