Daniel:Notebook/PosSequencing/2017-7-5

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Revision as of 23:18, 5 July 2017 by >Djacobse (→‎Results)
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emPCR 7-Strand Separation (Started Wednesday June 28)

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Protocol

  1. Template-bead binding; do this twice, once with no template
    1. Suspend 1 uL (10 ug, ~10M beads) beads in 100 uL Dynabuffer
    2. Apply magnet for 30 sec and remove supernatant
    3. Suspend in 20 uL Dynabuffer (conc. 2.5 ug/uL)
    4. Add 5 uL 500 nM (25 pmol total, 0.5X excess) dsDNA and 15 uL nfH2O per sample to bead solution; incubate at RT for 15 min; for NT sample add 20 dynabuffer/20 nfH2O
    5. Wash twice with 100 uL dynabuffer
    6. Resuspend beads in 50 uL TE buffer
  2. DRAQ5 staining
    1. Take 5 uL aliquots and add 43 uL nfH2O and 2 uL DRAQ5 (1:10000)
  3. Slide preparation
    1. Add 15 uL of aliquot to a slide
    2. Add a #1.5 coverslip and seal with nail polish; wait approximately 20 minutes before imaging

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