Daniel:Notebook/PosSequencing/2017-6-12

From ZhangLabWiki
Revision as of 23:59, 12 June 2017 by >Djacobse (→‎Protocol)
Jump to navigation Jump to search

Biotin vs Dual Biotin

Back to Calendar

Protocol

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
  5. qPCR
    1. Prepare 2 plates; 1 single biotin and 1 dual biotin
    2. For each sample prepare 58X master mix and (4X) master mix: 1X components set to 30 uL; add 2uL sample
    3. ol type="a">
    4. 58 (4) uL 1 uM primer2
    5. (4) uL 1 uM biotin/2biotin uracil primer
    6. 58 uL 10 pM barcode oligo v2
    7. 870 (60) uL 2X Kapa SYBR
    8. 638 (44) uL nfH2O
  6. Add 28 uL master mix according to plate (4X master mix is for positive controls, 58X is for samples and negative controls)
  7. Add 2 uL appropriate sample according to plate layout
  8. Use "standard" thermocycling program