Daniel:Notebook/PosSequencing/2017-6-12
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Biotin vs Dual Biotin
Protocol
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
- qPCR
- Prepare 2 plates; 1 single biotin and 1 dual biotin
- For each sample prepare 58X master mix and (4X) master mix: 1X components set to 30 uL; add 2uL sample
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- 58 (4) uL 1 uM primer2
- (4) uL 1 uM biotin/2biotin uracil primer
- 58 uL 10 pM barcode oligo v2
- 870 (60) uL 2X Kapa SYBR
- 638 (44) uL nfH2O
- Add 28 uL master mix according to plate (4X master mix is for positive controls, 58X is for samples and negative controls)
- Add 2 uL appropriate sample according to plate layout
- Use "standard" thermocycling program