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 ( | <li>Suspend 2 uL (20 ug) beads in 20 uL Dynabuffer (conc. 1 ug/uL)</li> | ||
<li>Add | <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
Protocol
This protocol is principally based off the Ion Torrent protocol for emulsion PCR
- 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 (20 ug) beads in 20 uL Dynabuffer (conc. 1 ug/uL)
- 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
- 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
- Mix 20 uL aqueous into 200 uL oil mixture
- Vortex for 30 seconds
- Repeat mixing to 100 uL (5 times)
- Mix with vortexer for 3 minutes
- Dilute with pure mineral oil for imaging (1:100)
- Emulsion PCR
- Emulsion Breaking
- Fluorescent Bead Binding
- Imaging