Daniel:Notebook/PosSequencing/2017-4-24: Difference between revisions

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<li>Template-Bead Binding</li>
<li>Template-Bead Binding</li>
<ol type="A">
<ol type="A">
<li>Suspend 2 uL (20 ug) beads in 100 uL Dynabuffer</li>
<li>Suspend 2.5 uL (25 ug) beads in 100 uL Dynabuffer</li>
<li>Apply magnet for 30 sec and remove supernatant</li>
<li>Apply magnet for 30 sec and remove supernatant</li>
<li>Suspend 2 uL (20 ug) beads in 20 uL Dynabuffer (conc. 1 ug/uL)</li>
<li>Suspend 2.5 uL (25 ug) beads in 20 uL Dynabuffer (conc. 1 ug/uL)</li>
<li>Add 10 uL 1 uM (10 pmol total, 10X excess) template oligo and 10 uL nfH<sub>2</sub>O per sample to bead solution; incubate at RT for 15 min</li>
<li>Add 10 uL 1 uM (10 pmol total, 10X excess) template oligo and 10 uL nfH<sub>2</sub>O per sample to bead solution; incubate at RT for 15 min</li>
<li>Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant</li>
<li>Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant</li>

Revision as of 23:07, 24 April 2017

Emulsion PCR

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Protocol

This protocol is principally based off the Ion Torrent protocol for emulsion PCR

  1. Oil Production
    1. Start from Monday April 14's oil prep (Span80-4.5%, Tween20-0.1%, TritonX100-0.05% in mineral oil)
  2. Template-Bead Binding
    1. Suspend 2.5 uL (25 ug) beads in 100 uL Dynabuffer
    2. Apply magnet for 30 sec and remove supernatant
    3. Suspend 2.5 uL (25 ug) beads in 20 uL Dynabuffer (conc. 1 ug/uL)
    4. Add 10 uL 1 uM (10 pmol total, 10X excess) template oligo and 10 uL nfH2O per sample to bead solution; incubate at RT for 15 min
    5. Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant
    6. Repeat wash step
    7. Resuspend beads in 30 uL TE buffer
    8. Mix together the three suspensions of beads with different oligos
  3. Oil-Aqueous Mixing
    1. Mix 20 uL aqueous into 200 uL oil mixture
    2. Vortex for 30 seconds
    3. Repeat mixing to 100 uL (5 times)
    4. Mix with vortexer for 3 minutes
    5. Dilute with pure mineral oil for imaging (1:100)
  4. Emulsion PCR
  5. Emulsion Breaking
  6. Fluorescent Bead Binding
  7. Imaging