Daniel:Notebook/PosSequencing/2017-6-12
Jump to navigation
Jump to search
Biotin vs Dual Biotin[edit]
Protocol[edit]
Experiment Map
File:ExperimentMap-20170610-Biotin-DualBiotin.png
- Template binding
- 28 samples-2 of each (Biotin/Dual biotin)
- Add 1 uL Dyanabeads (10 ug) to 100 uL Dynabuffer
- Pull down and resuspend in 20 uL dynabuffer
- Add 18 uL nfH2O and 2 uL 10 uM biotin or dual-biotin primer
- Incubate at RT for 15 minutes
- Remove supernatant and wash once with 100 uL Dynabuffer
- Controls
- Add 50 uL TE
- Incubate RT for 5 min
- Pull down, withdraw and save supernatant
- Repeat 2 more times (3 saved fractions)
- Add 50 uL TE for final suspension
- Heat Denaturation
- Add 50 uL TE
- Incubate 95C,90C, or 85C for 5 min
- Pull down, withdraw and save supernatant
- Repeat 2 more times (3 saved fractions)
- Add 50 uL TE for final suspension
- Heat Denaturation
- Add 50 uL alkaline buffer (12.0, 11.0, or 10.0)
- Pull down, withdraw and save supernatant
- Repeat 2 more times (3 saved fractions)
- Add 50 uL TE for final suspension
Continued tomorrow