Daniel:Notebook/PosSequencing/2017-7-14: Difference between revisions

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<li>Dye hybridization</li>
<li>Dye hybridization</li>
<ol type="A">
<ol type="A">
<li>Add 1 uL appropriate (10 uM) dye, A488-dc6 for sample 1 and Cy5-dc6 for sample 3</li>
<li>Resuspend beads in 30 uL 2X SSC</li>
<li>Incubate in the dark at 75C for 5 minutes</li>
<li>Mix 15 uL 4X SSC, 9 uL nfH<sub>2</sub>O, 3 uL 10 uM dcprobe6-A488, and 3 uL 10 uM dcprobe6-Cy5</li>
<li>Incubate at 55C for 30 minutes</li>
<li>Incubate master mix in the dark at 80C for 5 minutes</li>
<li>Incubate in the dark (RT) for 15 minutes</li>
<li>Add 10 uL master mix to each sample</li>
<li>Incubate at 55C for 30 minutes in the dark</li>
<li>Magnet pull down, remove supernatant and add 40 uL 2X SSC buffer</li>
<li>Magnet pull down, remove supernatant and add 40 uL 2X SSC buffer</li>
<li>Add 15 uL to a slide for analysis</li>
<li>Add 15 uL to a slide for analysis</li>

Revision as of 17:56, 14 July 2017

emPCR 9-Concentration Comparison (Started Wednesday)

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Protocol-Part 3

  1. Dye hybridization
    1. Resuspend beads in 30 uL 2X SSC
    2. Mix 15 uL 4X SSC, 9 uL nfH2O, 3 uL 10 uM dcprobe6-A488, and 3 uL 10 uM dcprobe6-Cy5
    3. Incubate master mix in the dark at 80C for 5 minutes
    4. Add 10 uL master mix to each sample
    5. Incubate at 55C for 30 minutes in the dark
    6. Magnet pull down, remove supernatant and add 40 uL 2X SSC buffer
    7. Add 15 uL to a slide for analysis
    8. Seal coverslip with nail polish and wait approximately 30 minutes to dry