Daniel:Notebook/PosSequencing/2017-5-20: Difference between revisions

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>Djacobse
>Djacobse
 
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<li>Add 2 uL samples and 1 (or 2) uL primer according to plate layout</li>
<li>Add 2 uL samples and 1 (or 2) uL primer according to plate layout</li>


[[File:PlateLayout-20170520-SinglePrimer.png|400px]]
[[File:PlateLayout-20170520-SinglePrimer.png]]


<li>qPCR Cycles</li>
<li>qPCR Cycles</li>

Latest revision as of 15:18, 25 May 2017

Single Stranded Probe Binding Test (Started Yesterday)[edit]

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Protocol[edit]

  1. Single primer qPCR
    1. Make the following master mixes
    2. Reagent uL in Single Rxn Master Mix P2 (3.2X) Master Mix BioU (3.2X) Master Mix Dual (2.2X)
      Primer 2 (10 uM) 1 3.2 3.2 2.2
      BioU Primer (10 uM) 1 0 0 2.2
      2X Kapa SYBR Master Mix 20 64 64 44
      nfH2O 17 54.4 54.4 35.2
      Total   121.6 121.6 83.6
    3. Add 37 uL or 38 uL (dual) to the appropriate lanes
    4. Add 2 uL samples and 1 (or 2) uL primer according to plate layout
    5. File:PlateLayout-20170520-SinglePrimer.png
    6. qPCR Cycles
      1. 95C 3 min
      2. 95C 3 sec
      3. 55C 30 sec
      4. 72C 20 sec
      5. plate read
      6. goto b x30
      7. 72C 2 min
      8. 16C hold
  2. Bead Binding
    1. Suspend 1 uL (10 ug, ~5M beads designed at 2.5 pmol) beads in 100 uL Dynabuffer
    2. Apply magnet for 30 sec and remove supernatant
    3. Suspend in 15 uL Dynabuffer (conc. 1 ug/uL)
    4. Add 15 uL barcode oligo; incubate at RT for 15 min
    5. Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant
    6. Add 1 uL appropriate dye probe in 49 uL SSC incubate in the dark (RT) for 15 minutes;
    7. Add 15 uL to a slide for analysis
    8. Remove the rest of the supernatant after bead pulldown