Daniel:Notebook/PosSequencing/2017-6-26
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Beadless emPCR (Started Thursday 6/22/17)[edit]
Since the results on Friday showed viable product (at least in the 100 nM sample), I'm going to finish up that experiment with strand denaturation and single primer qPCR.
Protocol[edit]
- Strand Separation (DNA Denaturation)
- Add 500 uL KOH buffer (pH 12.0)
- Spin down sample at 13,000rpm for 1 minute
- Remove all but 40uL buffer
- Repeat KOH steps A-C once more
- Add 500 uL 4X SSC buffer (pH 12.0)
- Spin down sample at 13,000rpm for 1 minute
- Remove all but 10uL buffer
- Resuspend in 80 uL 2X SSC buffer
- Single primer qPCR
- Make the following master mixes
- Aliquot 35 uL master mix to appropriate lanes
- Add 2 uL sample according to plate layout below File:PlateLayout-Beadless-emPCR-singleprimer-20170626.png
- Run the following thermocycler program
- 95C 3 min
- 95C 3 sec
- 45C 30 sec
- 72C 20 sec
- plate read
- goto b x90
- 72C 2 min
- 16C hold
Reagent | uL in Single Rxn | Master Mix P2 (4.2X) | Master Mix BioU (4.2X) | Master Mix Dual (6.2X) |
Primer 2 (10 uM) | 1 | 4.2 | 0 | 6.2 |
BioU Primer (10 uM) | 1 | 0 | 4.2 | 6.2 |
2X Kapa SYBR Master Mix | 20 | 84 | 84 | 124 |
nfH2O | 16 | 67.2 | 67.2 | 93 |
Total* (+ 5 uL sample=40) | 35 | 155.4 | 155.4 | 229.4 |
Results[edit]
- PlateLayout-Beadless-emPCR-singleprimer-20170626.png
Plate layout
- Beadless-emPCR-singleprimer-20170626-plateCTs-normed.png
Plate CTs
- Beadless-emPCR-singleprimer-20170626-rawcurves.png
Raw curves
emPCR 6[edit]
Based on the results from Friday and continued above, I think this means I'm clear to try again on emPCR using the lower annealing temp. I'll give 15 more cycles since the pM sample didn't amplify.
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 (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
- 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, 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