Daniel:Notebook/PosSequencing/2017-7-6
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emPCR8[edit]
For this one, I'm going to do the previous protocol, but using lambda exonuclease for digestion to reduce the complexity of the experiment. Finding proper denaturation conditions will take extra time that we don't really need to use since lambda is not too expensive and serves well for our proof of concept.
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 3 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 (25 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
- Add 83 uL of master mix to each set of 10 uL beads
- Add barcode oligos and water mix according to table
- Add 2 uL DreamTaq Polymerase to each sample
- 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
- 45C 30 sec
- 70C 30 sec
- Goto b 95 times
- 4C forever
Component | uL Added | 3.2X Master Mix uL Added | Final Concentration |
DreamTaq 10X PCR Buffer (20 mM MgCl2) | 10 | 32 | 1X |
10 mM dNTPs | 10 | 32 | 1 mM |
25 mM MgCl2 | 6 | 19.2 | 3.5 mM |
10 uM Primer A (Free primer-Primer 2) | 10 | 32 | 1 uM |
10 uM dual biotin primer | 0.5 | 1.6 | 0.1 uM |
Hydrogel beads, 2M/uL | 10 | 0 | 25 M |
DreamTaq Polymerase (5 U/uL) * | 2 | 0 | 10 Units |
Barcode Oligo v1 or v3 (template, 100 pM) | 5 | 0 | 10 pM |
100 mM Ammonium Sulfate | 5 | 16 | 5 mM |
1 mM Spermidine*** | 10 | 32 | 100 uM |
TIPP | 0 | 0 | 0 |
Water | 26.5 | 84.8 | NA |
Total Volume | 95 | 249.6 |
Sample 1 | Sample 2 | Sample 3 | |
Barcode Oligo v1 (100 pM) | 5 | 0 | 5 |
Barcode Oligo v3 (100 pM) | 0 | 5 | 5 |
nfH2O | 5 | 5 | 0 |
******
Continued tomorrow