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

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<li>Mix with vortexer for 3 minutes</li>
<li>Mix with vortexer for 3 minutes</li>
<li>Dilute with pure mineral oil for imaging (1:100)</li>
<li>Dilute with pure mineral oil for imaging (1:100)</li>
<li>Distribute 300 uL total volume into 6 tubes, 50 uL each</li>
</ol>
</ol>
<li>Emulsion PCR</li>
<li>Emulsion PCR</li>

Revision as of 00:27, 25 April 2017

Emulsion PCR

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Protocol

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

  1. Template DNA Production (Barcode Oligos)
    1. Start with oligo concentrations of 100 uM (100 pmol/uL)
    2. Make dilutions by adding 5 uL into 50 uL (1:10 dilutions)
    3. Dilute 8X times (final concentration 1 pM or 1 amol/uL)
    4. Make oligo solution by adding 5 uL barcode1 (Alexa488) and barcode3(Cy5) since they have low overlap-Final concentration of oligo is 1 pM; adding 10 uL to PCR mix makes it 0.1pM, the suggested concentration from Ion Torrent protocol
  2. Oil Production
    1. Start from Monday April 14's oil prep (Span80-4.5%, Tween20-0.1%, TritonX100-0.05% in mineral oil)
  3. Template-Bead Binding
    1. Suspend 2.5 uL (25 ug) beads in 100 uL Dynabuffer
    2. Apply magnet for 30 sec and remove supernatant
    3. Suspend 2.5 uL (25 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 5 uL TE buffer
    8. Mix together the three suspensions of beads with different oligos
    9. Prepare the aqueous mix according to the table below
    10. Mix ingredients using the following table (see Notes)
    11. Component Ion Torrent Concentration My uL Added My Final Concentration
      5X PCR Buffer (Taq) * 1X 20 1X
      10 mM dNTPs ** 2.5 mM 25 2.5 mM
      25 mM MgCl2 3.5 mM 14 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
      Taq? Polymerase (5 U/uL) * 90 Units 10  
      DNA template 2nd Dilution (Barcode Oligos) 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 0 0
      TIPP NA 0 0
      Water NA 5 NA
      Total Volume   100  
  4. 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)
    6. Distribute 300 uL total volume into 6 tubes, 50 uL each
  5. Emulsion PCR
    1. Perform PCR with the following settings
      1. 95C 2 min
      2. 95C 20 sec
      3. 58C 30 sec
      4. 68C 30 sec
      5. Goto b 40 times
      6. 95C 30 sec
      7. 68C 6 min
      8. Goto f 20X times
      9. 4C forever

    Protocol Part 2

    1. Emulsion Breaking
      1. With a 200 uL pipette pool the emulsion into a 1.5 mL tube; save the tip and put aside
      2. Add 100 uL isopropyl alcohol (IPA) to tube with 200 uL tip (use new tip)
      3. Draw up IPA with tip from part A, mix to resuspend tip
      4. Vortex the pooled sample in the 1.5 mL tube for 30 seconds
      5. Spin down the sample 13,000 rpm for 5 minutes
      6. Take off oil-IPA mix (top layer) but do not disrupt the oil/aqueous interface
      7. Add 1000 uL IPA and vortex for 30 seconds
      8. Spin down sample at 13,000rpm for 3 minutes
      9. Again remove IPA without disturbing the pellet
      10. Add 500 uL 4X SSC wash buffer to 1.5 mL tube and vortex for 30 seconds
      11. Go back and mix thoroughly by pipetting up and down; the pellet may be hard to resuspend
      12. Leave on 1.5mL mixer at 1300rpm 1 minute
      13. Spin down sample at 13,000rpm for 1 minute
      14. Remove all but 40uL wash buffer
      15. Repeat steps 11 to 15 two times (j to o)
      16. Resuspend in ~100 uL 4X SSC buffer
    2. Fluorescent Bead Binding
    3. Imaging

    Notes

    • "*": NEB Taq has terrible information. Since the ThermoFisher Taq is much better documented and available at a reasonable price, I will go with that in the future. It is on the order sheet and I will switch to that in the future. Since I'm not running the protocol today (It's almost 5), the Thermo
    • "**": dNTPs are also available at up to 100 mM for each dNTP if this concentration proves too annoying to work with
    • "***": Spermidine seems useful so I'm ordering it. We may actually have Ammonium sulfate but I'm not sure it's necessary