Daniel:Notebook/PosSequencing/2017-5-25
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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
Component | Ion Torrent Concentration | My uL Added- Sample (Single) | My Final Concentration |
DreamTaq 10X PCR Buffer (20 mM MgCl2) | 1X | 10 | 1X |
10 mM dNTPs | 2.5 mM | 25 | 2.5 mM |
25 mM MgCl2 | 3.5 mM | 6 | 3.5 mM |
100 uM Primer A (Free primer-Primer 2) | 9 uM | 5 | 5 uM |
10 uM Primer B (Bound primer-Primer 1) | 0.1 uM | 1 | 0.1 uM |
Hydrogel beads, 2M/uL | 50 Million | 10 | 60 M |
DreamTaq Polymerase (5 U/uL) * | 90 Units | 18 | 90 Units |
DNA template 2nd Dilution (Barcode Oligos 1 pM) | 0.12 pM | 10 | 0.012 fmol total (0.006 fmol each?) |
1 M Ammonium Sulfate | 5 mM | 0 | 0 |
1 mM Spermidine*** | 100 uM | 10 | 0 |
TIPP | NA | 0 | 0 |
Water | NA | 5 | NA |
Total Volume | 100 |