Daniel:Notebook/PosSequencing/2017-6-28
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emPCR 7-Strand Separation
Since yesterday's results showed DNA in the two fractions (10 nM and 10 pM) after emulsion breaking but before adding the alkaline buffer for strand separation. Therefore, I'll try again but I'll split the fractions post-emulsion breaking and use several different pH values to test effectiveness.
Protocol
- 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 (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
- Note: The 1 pM sample is the concentration suggested to use. This will help me know about how many cycles to use for production in the future
- 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 95 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, 1 pM*) | 5 | 5* | 10 pM, 0.1 pM* |
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 |