Daniel:Notebook/PosSequencing/2017-6-21
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Bead qPCR Test
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
Beads in Sample | Dilution (Power 10) | Amount Added (uL) |
10 M | 0 | 2 |
2 M | 1 | 4 |
1 M | 1 | 2 |
0.2 M | 2 | 4 |
0.1 M | 2 | 2 |
20 K | 3 | 4 |
10 K | 3 | 2 |
1 K | 4 | 2 |