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

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


<ol>
<ol>
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<li>Suspend 2 uL (20 ug) beads in 100 uL Dynabuffer</li>
<li>Suspend 2 uL (20 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 (100 ug) beads in 20 uL Dynabuffer (conc. 5 ug/uL)</li>
<li>Suspend 2 uL (20 ug) beads in 20 uL Dynabuffer (conc. 1 ug/uL)</li>
<li>Add 20 uL 10 uM template oligo 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>
<li>Repeat wash step</li>
<li>Repeat wash step</li>

Revision as of 21:04, 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 uL (20 ug) beads in 100 uL Dynabuffer
    2. Apply magnet for 30 sec and remove supernatant
    3. Suspend 2 uL (20 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