Daniel:Notebook/PosSequencing/2017-6-21
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Bead qPCR Test
This experiment has 2 purposes: to determine the density of beads necessary to optically block qPCR detection (and hopefully some levels that don't) as well as the efficiency of bead-based qPCR vs aqueous. This will hopefully help me make the next experiment run smoother, specifically using digital qPCR to amplify the beads.
Protocol
- Template-Bead Binding; Make 4 samples and follow this protocol for each
- Suspend 5 uL (50 ug, ~50M beads) beads in 100 uL Dynabuffer
- Apply magnet for 30 sec and remove supernatant
- Suspend in 20 uL Dynabuffer (conc. 2.5 ug/uL)
- Add 5 uL 10 uM (50 pmol total, 2X excess) dual biotin oligo and 15 uL nfH2O per sample to bead solution; incubate at RT for 15 min
- Wash twice with 100 uL Dynabuffer
- Resuspend beads in 10 uL TE buffer
- Dilutions
- Make dilutions using 2 uL suspended beads with 18 uL TE for dilutions
- Make dilutions of 1:10, 1:100, 1:1000, and 1:10000 Sample Matrix
- qPCR
- Make the following master mix
- Add 36 uL master mix to each lane
- Add n uL of sample and 4-n uL nfH2O to appropriate lanes according to plate layout
- For the "Supernatant" sample add 10 uL TE to original beads (0 dilution), mix to suspend, then pull down; take 4 uL supernatant for sample File:PlateLayout-BeadsqPCR-20170621.png
- Vortex and spin down after sealing wells
- Use the standard thermocycler program->40 cycles
Beads in Sample | Dilution (Power 10) | Amount Added (uL) | ~Amt BioU Primer (pmol) |
10 M | 0 | 2 | 5 pmol |
2 M | 1 | 4 | 1 pmol |
1 M | 1 | 2 | 500 nmol |
0.2 M | 2 | 4 | 100 nmol |
0.1 M | 2 | 2 | 50 nmol |
20 K | 3 | 4 | 10 nmol |
10 K | 3 | 2 | 5 nmol |
1 K | 4 | 2 | 1 nmol |
Reagent | 1X Added | Master Mix (21.2X) |
Primer 2 (10 uM) | 1 | 21.2 |
Barcode Oligo 100 pM | 1 | 21.2 (1) |
Beads | 4 | 84.8 |
Kapa 2X SYBR Fast Mix | 20 | 424 |
nfH2O | 14 | 296.8 (155.4) |
Total | 40 | 848 |
Results
- PlateLayout-BeadsqPCR-20170621.png
Plate layout
- BeadqPCR-plateCTs-normed.png
Plate CTs
- BeadqPCR-rawcurves.png
Raw curves
- BeadqPCRTest-20170621.png
Cycle threshold vs. input (log pmol)
Discussion
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