Daniel:Notebook/PosSequencing/2017-5-22: Difference between revisions

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>Djacobse
>Djacobse
Line 14: Line 14:
<li>Add 15 uL to a slide for analysis</li>
<li>Add 15 uL to a slide for analysis</li>
<li>Seal coverslip with nail polish and wait approximately 30 minutes to dry</li>
<li>Seal coverslip with nail polish and wait approximately 30 minutes to dry</li>
<li>Image on microscope</li>
</ol>
<li>Imaging Parameters</li>
<ol type="A">
<li>Confocal Microscope</li>
<li>488 Laser-3.00%</li>
<ol type="a">
<li>PMT Trans-Gain 225V
<li>PMT1-Range:500-550-Gain 550V</li>
</ol>
<li>642 Laser-8.00%</li>
<ol type="a">
<li>PMT Trans-Gain 300V
<li>PMT1-Range:500-550-Gain 6000V</li>
<li>PMT3-Range:750-800-Gain 550V</li></ol>
</ol></ol>
</ol></ol>



Revision as of 17:40, 22 May 2017

Single Stranded Probe Binding Test (Started Friday May 19)

Back to Calendar

Protocol (Continued from Saturday May 20)

  1. Dye hybridization
    1. Mix 20 uL ssDNA (lambda digested) and 20 uL dsDNA (original, non-digested template)
    2. Add 1 uL appropriate (10 uM) dye, A488-dc6 for sample 1 and Cy5-dc6 for sample 3
    3. Incubate in the dark (RT) for 15 minutes
    4. Magnet pulldown, remove supernatant and add 40 uL 2X SSC buffer
    5. Add 15 uL to a slide for analysis
    6. Seal coverslip with nail polish and wait approximately 30 minutes to dry
  2. Imaging Parameters
    1. Confocal Microscope
    2. 488 Laser-3.00%
      1. PMT Trans-Gain 225V
      2. PMT1-Range:500-550-Gain 550V
    3. 642 Laser-8.00%
      1. PMT Trans-Gain 300V
      2. PMT1-Range:500-550-Gain 6000V
      3. PMT3-Range:750-800-Gain 550V

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