Daniel:Notebook/PosSequencing/2017-5-25: Difference between revisions
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>Djacobse (Created page with "=Emulsion PCR Test 2= Back to Calendar From the results on ==Protocol== <li>Oil Production</li> <ol type="A"> <li>Start from Daniel:No...") |
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==Protocol== | ==Protocol== | ||
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Revision as of 16:12, 25 May 2017
Emulsion PCR Test 2
From the results on
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; Make 4 samples and follow this protocol for each
- Suspend 5 uL (50 ug, ~50M beads) beads in 100 uL Dynabuffer
- Apply magnet for 30 sec and remove supernatant
- Suspend in 20 uL Dynabuffer (conc. 2.5 ug/uL)
- Add 2 uL 1 uM (10 pmol total, 2X excess) template oligo and 18 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 10 uL TE buffer
- Mix together the three suspensions of beads with different oligos
- Mix ingredients using the following table
- Aliquot 90 uL master mix into 4 tubes
- Add 10 uL appropriate primers according to sample table
- Aliquot 50 uL each non-emulsion sample into new tubes (A/B)
- 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)
- Distribute 300 uL total volume into 6 tubes, 50 uL each; use same pipette tip; only made 5 (oil sticks to tip)
- Emulsion PCR
- Perform PCR with the following settings
- 95C 2 min
- 95C 20 sec
- 58C 30 sec
- 70C 30 sec
- Goto b 40 times
- 95C 30 sec
- 68C 3 min
- Goto f 20X times
- 4C forever