Daniel:Notebook/PosSequencing/2017-6-12: Difference between revisions
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>Djacobse (Created page with "=Biotin vs Dual Biotin= Back to Calendar ==Protocol== '''Experiment Map''' Image:ExperimentMap-20170610-Biotin-DualBiotin.png|700x525px...") |
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<li>Heat Denaturation</li> | <li>Heat Denaturation</li> | ||
<ol type="A"> | <ol type="A"> | ||
<li>Add 50 uL alkaline buffer ( | <li>Add 50 uL alkaline buffer (12.0, 11.0, or 10.0)</li> | ||
<li>Pull down, withdraw and save supernatant</li> | <li>Pull down, withdraw and save supernatant</li> | ||
<li>Repeat 2 more times (3 saved fractions)</li> | <li>Repeat 2 more times (3 saved fractions)</li> | ||
Line 45: | Line 45: | ||
<li>qPCR</li> | <li>qPCR</li> | ||
<ol type="A"> | <ol type="A"> | ||
<li></li> | <li>Prepare 2 plates; 1 single biotin and 1 dual biotin</li> | ||
<li>For each sample prepare 58X master mix and (4X) master mix: 1X components set to 30 uL; add 2uL sample</li> | |||
<li>ol type="a"> | |||
<li>58 (4) uL 1 uM primer2</li> | |||
<li>(4) uL 1 uM biotin/2biotin uracil primer</li> | |||
<li>58 uL 10 pM barcode oligo v2</li> | |||
<li>870 (60) uL 2X Kapa SYBR</li> | |||
<li>638 (44) uL nfH<sub>2</sub>O</li></ol> | |||
<li>Add 28 uL master mix according to plate (4X master mix is for positive controls, 58X is for samples and negative controls)</li> | |||
<li>Add 2 uL appropriate sample according to plate layout</li> | |||
<li>Use "standard" thermocycling program</li> | |||
</ol> | </ol> | ||
</ol> | </ol> |
Revision as of 23:59, 12 June 2017
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
- ol type="a">
- 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