Daniel:Notebook/PosSequencing/2017-6-6

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Revision as of 18:30, 9 June 2017 by >Djacobse (→‎Results)
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Aqueous PCR 2

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Last time I may have been using too much enzyme. Too high of glycerol can inhibit PCR, and

Protocol

  1. Template-Bead Binding
    1. Suspend 12 uL (120 ug, ~120M 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 2.5 uL 100 uM Biotin-Uracil primer1 (250 pmol total, 2X excess) and 17.5 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 30 uL TE buffer
  2. qPCR
    1. Mix together the three suspensions of beads with different oligos
    2. Mix ingredients using the following table
    3. Component IT Concentration Sample (Single) Sample Concentration No SYBR Overloaded 10 Units NTC 5.2X Master Mix Master Mix Applicable
      DreamTaq 10X PCR Buffer (20 mM MgCl2) 1X 5 1X 5 5 5 5 26 1
      10 mM dNTPs 2.5 mM 12.5 2.5 mM 12.5 12.5 12.5 12.5 65 1
      25 mM MgCl2 3.5 mM 3 3.5 mM 3 3 3 3 15.6 1
      100 uM Primer A (Free primer-Primer 2) 9 uM 2.5 5 uM 2.5 2.5 2.5 2.5 13 1
      10 uM Primer B (Bound primer-Primer Biotin-Uracil) 0.1 uM 0.5 0.1 uM 0.5 0.5 0.5 0.5 2.6 1
      Hydrogel beads, 2M/uL 50 Million 5 60 M 5 5 5 5 26 1
      DreamTaq Polymerase (5 U/uL) * 90 Units/150 uL 6 30 Units 6 9 2 6 0 0
      DNA template 2nd Dilution (Barcode Oligo v2 1 pM) 0.12 pM 5 0.01 fmol 5 5 5 0 0 0
      1 M Ammonium Sulfate 5 mM 0 0 0 0 0 0 0 1
      1 mM Spermidine*** 100 uM 5 0 5 5 5 5 26 1
      TIPP NA 0 0 0 0 0 0 0 1
      Water NA 4.5 NA 5.5 1.5 8.5 9.5 0 0
      SYBR Green (100:1) NA 1 NA 0 1 1 1 0 0
      Total Volume   50   50 50 50 50 174.2  
    4. Add 33.5 uL master mix to each lane
    5. Add Template, Taq, and nfH2O as relevant and dictated by table/plate layout
    6. File:PlateLayout-20170606-aqPCR2 .png
    7. 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

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