Daniel:Notebook/PosSequencing/2017-6-19
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emPCR Test 5[edit]
Check the results from last week. The post-emulsion breaking sample showed moderate amplification over the dual NTC. This means that it might just need a few more cycles. I'll try again, as well as a high concentration sample. This one should be closer to a positive control, since starting that high should need little to no amplification to fill out the beads.
Protocol[edit]
- Oil phase
- Mix 5 uL Triton X, 40 uL Tween-80, and 450 uL Span 80 into 2 mL Mineral oil and 8 mL Tegosoft DEC
- Vortex vigorously to mix
- Template-Bead Binding; Make 4 samples and follow this protocol for each
- Suspend 2.5 uL (25 ug, ~25M 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
- Aqueous Phase Preparation
- Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant
- Repeat wash step
- Resuspend beads in 10 uL TE buffer
- Mix ingredients using the following table
- Oil-Aqueous Mixing
- Mix 10 uL aqueous into 100 uL oil mixture
- Vortex for 30 seconds
- Repeat mixing to 100 uL (5 times)
- Mix with vortexer for 3 minutes
- Dilute with pure mineral oil for imaging (1:100)
- Distribute 150 uL total volume into 3 tubes, 50 uL each; use same pipette tip
- Emulsion PCR
- Perform PCR with the following settings
- 95C 2 min
- 95C 20 sec
- 58C 30 sec
- 70C 30 sec
- Goto b 80 times
- 4C forever
Component | uL Added | uL Added | Final Concentration |
DreamTaq 10X PCR Buffer (20 mM MgCl2) | 10 | 10 | 1X |
10 mM dNTPs | 10 | 10 | 1 mM |
25 mM MgCl2 | 6 | 6 | 3.5 mM |
10 uM Primer A (Free primer-Primer 2) | 10 | 10 | 1 uM |
10 uM dual biotin primer | 0.5 | 0.5 | 0.1 uM |
Hydrogel beads, 2M/uL | 10 | 10 | 25 M |
DreamTaq Polymerase (5 U/uL) * | 2 | 2 | 10 Units |
Barcode Oligo v1 (template, 100 pM, 100 nM) | 5 | 5* | 10 pM, 10 nM* |
100 mM Ammonium Sulfate | 5 | 5 | 5 mM |
1 mM Spermidine*** | 10 | 10 | 100 uM |
TIPP | 0 | 0 | 0 |
Water | 36.5 | 36.5 | NA |
Total Volume | 100 | 100 |
Continued tomorrow