Daniel:Notebook/PosSequencing/2017-5-25

From ZhangLabWiki
Revision as of 16:21, 25 May 2017 by >Djacobse (→‎Protocol)
Jump to navigation Jump to search

Emulsion PCR Test 2

Back to Calendar

From the results on

Protocol

  1. Oil Production
    1. Start from Monday April 14's oil prep (Span80-4.5%, Tween20-0.1%, TritonX100-0.05% in mineral oil)
  2. Template-Bead Binding; Make 4 samples and follow this protocol for each
    1. Suspend 5 uL (50 ug, ~50M beads) beads in 100 uL Dynabuffer
    2. Apply magnet for 30 sec and remove supernatant
    3. Suspend in 20 uL Dynabuffer (conc. 2.5 ug/uL)
    4. 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
    5. Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant
    6. Repeat wash step
    7. Resuspend beads in 10 uL TE buffer
    8. Mix together the three suspensions of beads with different oligos
    9. Mix ingredients using the following table
    10. 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  
    11. Aliquot 90 uL master mix into 4 tubes
    12. Add 10 uL appropriate primers according to sample table
    13. Aliquot 50 uL each non-emulsion sample into new tubes (A/B)
  3. 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; use same pipette tip; only made 5 (oil sticks to tip)
  4. Emulsion PCR
    1. Perform PCR with the following settings
      1. 95C 2 min
      2. 95C 20 sec
      3. 58C 30 sec
      4. 70C 30 sec
      5. Goto b 40 times
      6. 95C 30 sec
      7. 68C 3 min
      8. Goto f 20X times
      9. 4C forever