Daniel:Notebook/PosSequencing/2017-6-12

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Biotin vs Dual Biotin[edit]

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Protocol[edit]

Experiment Map

File:ExperimentMap-20170610-Biotin-DualBiotin.png


  1. Template binding
    1. 28 samples-2 of each (Biotin/Dual biotin)
    2. Add 1 uL Dyanabeads (10 ug) to 100 uL Dynabuffer
    3. Pull down and resuspend in 20 uL dynabuffer
    4. Add 18 uL nfH2O and 2 uL 10 uM biotin or dual-biotin primer
    5. Incubate at RT for 15 minutes
    6. Remove supernatant and wash once with 100 uL Dynabuffer
  2. Controls
    1. Add 50 uL TE
    2. Incubate RT for 5 min
    3. Pull down, withdraw and save supernatant
    4. Repeat 2 more times (3 saved fractions)
    5. Add 50 uL TE for final suspension
  3. Heat Denaturation
    1. Add 50 uL TE
    2. Incubate 95C,90C, or 85C for 5 min
    3. Pull down, withdraw and save supernatant
    4. Repeat 2 more times (3 saved fractions)
    5. Add 50 uL TE for final suspension
  4. Heat Denaturation
    1. Add 50 uL alkaline buffer (12.0, 11.0, or 10.0)
    2. Pull down, withdraw and save supernatant
    3. Repeat 2 more times (3 saved fractions)
    4. Add 50 uL TE for final suspension

Continued tomorrow