Daniel:Notebook/PosSequencing/2017-4-24: Difference between revisions
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<ol> | <ol> | ||
<li>Oil Production</li> | |||
<ol type="A"> | |||
<li>Start from [[Daniel:Notebook/PosSequencing/2017-4-10|Monday April 14]]'s oil prep (Span80-4.5%, Tween20-0.1%, TritonX100-0.05% in mineral oil)</li></ol> | |||
<li>Template-Bead Binding</li> | <li>Template-Bead Binding</li> | ||
<ol type="A"> | |||
<li>Suspend 2 uL (20 ug) beads in 100 uL Dynabuffer</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>Add 20 uL 10 uM template oligo 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>Repeat wash step</li> | |||
<li>Resuspend beads in 30 uL TE buffer</li> | |||
<li>Mix together the three suspensions of beads with different oligos</li> | |||
</ol> | |||
<li>Oil-Aqueous Mixing</li> | <li>Oil-Aqueous Mixing</li> | ||
<li>Emulsion PCR</li> | <li>Emulsion PCR</li> |
Revision as of 18:00, 24 April 2017
Emulsion PCR
Protocol
- Oil Production
- Start from Monday April 14's oil prep (Span80-4.5%, Tween20-0.1%, TritonX100-0.05% in mineral oil)
- Template-Bead Binding
- Suspend 2 uL (20 ug) beads in 100 uL Dynabuffer
- Apply magnet for 30 sec and remove supernatant
- Suspend 2 uL (100 ug) beads in 20 uL Dynabuffer (conc. 5 ug/uL)
- Add 20 uL 10 uM template oligo per sample to bead solution; incubate at RT for 15 min
- Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant
- Repeat wash step
- Resuspend beads in 30 uL TE buffer
- Mix together the three suspensions of beads with different oligos
- Oil-Aqueous Mixing
- Emulsion PCR
- Fluorescent Bead Binding
- Imaging