Daniel:Notebook/PosSequencing/2017-5-20: Difference between revisions
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>Djacobse (Created page with "=Single Stranded Probe Binding Test (Started Yesterday)= Back to Calendar ==Protocol== <ol sta...") |
>Djacobse |
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<li>72C 2 min</li> | <li>72C 2 min</li> | ||
<li>16C hold</li> | <li>16C hold</li> | ||
</ol></ol></li> | </ol></ol> | ||
<ol> | |||
<li>Bead Binding</li> | |||
<ol type="A"> | |||
<li>Suspend 1 uL (10 ug, ~5M beads designed at 2.5 pmol) beads in 100 uL Dynabuffer</li> | |||
<li>Apply magnet for 30 sec and remove supernatant</li> | |||
<li>Suspend in 15 uL Dynabuffer (conc. 1 ug/uL)</li> | |||
<li>Add 15 uL barcode oligo; incubate at RT for 15 min</li> | |||
<li>Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant</li> | |||
<li>Add 1 uL appropriate dye probe in 49 uL SSC incubate in the dark (RT) for 15 minutes;</li> | |||
<li>Add 15 uL to a slide for analysis</li> | |||
<li>Remove the rest of the supernatant after bead pulldown</li> | |||
</ol> |
Revision as of 21:18, 20 May 2017
Single Stranded Probe Binding Test (Started Yesterday)
Protocol
- Single primer qPCR
- Make the following master mixes
- Add 37 uL or 38 uL (dual) to the appropriate lanes
- Add 2 uL samples and 1 (or 2) uL primer according to plate layout File:PlateLayout-20170520-SinglePrimer.png
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x30
- 72C 2 min
- 16C hold
- Bead Binding
- Suspend 1 uL (10 ug, ~5M beads designed at 2.5 pmol) beads in 100 uL Dynabuffer
- Apply magnet for 30 sec and remove supernatant
- Suspend in 15 uL Dynabuffer (conc. 1 ug/uL)
- Add 15 uL barcode oligo; incubate at RT for 15 min
- Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant
- Add 1 uL appropriate dye probe in 49 uL SSC incubate in the dark (RT) for 15 minutes;
- Add 15 uL to a slide for analysis
- Remove the rest of the supernatant after bead pulldown
Reagent | uL in Single Rxn | Master Mix P2 (3.2X) | Master Mix BioU (3.2X) | Master Mix Dual (2.2X) |
Primer 2 (10 uM) | 1 | 3.2 | 3.2 | 2.2 |
BioU Primer (10 uM) | 1 | 0 | 0 | 2.2 |
2X Kapa SYBR Master Mix | 20 | 64 | 64 | 44 |
nfH2O | 17 | 54.4 | 54.4 | 35.2 |
Total | 121.6 | 121.6 | 83.6 |