Daniel:Notebook/ComboLock/2016-12-16
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Overnight Capture (Started Yesterday)[edit]
Protocol[edit]
- Circularization
- Prepare 7X uL Phusion mix
- Add 20 uL Phusion mix to 20 uL of reaction
- Incubate at 60C for 2 hours
- Exonuclease Digestion
- Mix 12.5 uL Exo I (20U/uL) and 2.5 uL ExoIII (100U/uL)
- Add 2 uL to each sample, mix by swirling pipette tip
- Incubate for 1 hour at 37C
- Heat kill by incubating for 5 min at 95C
- Rolling Circle Amplification
- Prepare 7X Master mix according to table below
- Add 15 uL master mix to each tube
- Add sample to new reaction tubes according to following table; use template from both supernatant only and beads
- Incubate at 37C for 3 hours
- Incubate at 65C for 10 minutes;Hold at 10C until next step
- qPCR
- Make master mix according to following recipe
- 151.2 uL nfH2O
- 180 uL SYBR Fast
- 7.2 uL 10 uM AmpF
- Add 47 uL master mix to each well
- Add 2 uL sample and 1 uL appropriate Reverse Primer (1 uM) according to table
- 2016-12-16-MirrorCProbe-ONCap-Supernatant.png
Gel image-supernatant RCA
- 2016-12-16-MirrorCProbe-ONCap-Beads.png
Gel image-Beads RCA
- qPCR
- Make master mix according to following recipes
- Add 48 uL master mix to each well
- Add 2 uL sample according to table in the following section
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x24
- 72C 2 min
- 16C hold
- TBE Gel
- Mix 144 uL TBE, 36 uL 6x loading dye
- Aliquot 10 uL per sample/ladder lane onto parafilm
- Add 2 uL of sample or ladder to correct drop
- Load 10 uL in to well
- Run gel for 22 minutes at 250V
- Open gel and stain with 3 uL SYBR Gold for 3 minutes
- Rinse gel and image in gel doc
- 2016-12-16-MirrorCProbe-ONCap-Adaptors-CProbes.png
Gel image 1-Sample C probe detection
- 2016-12-16-MirrorCProbe-ONCap-Adaptors-Controls.png
Gel image 2-Controls gel
Reagent | Stock Conc | Final Amount | 1x Vol (uL) | MM Vol (7x) (uL) |
NAD+ | 5 mM | 40 nmol | 8 | 56 |
dNTP | 1 mM | 600 pmol | 0.6 | 4.2 |
Betaine | 5 M | 15 umol | 3 | 21 |
10X AmpLigase Buffer | 10X | 1X | 2 | 14 |
Amp Ligase | 5 U/uL | 10 U | 2 | 14 |
Phusion HF DNA Polymerase | 2000 U/mL | 6.4U | 3.2 | 22.4 |
nf H2O | 1.2 | 8.4 | ||
Total | 20 | 140 |
Reagent | uL Added | Master Mix (13X) |
Template | 5 | NA |
RCA Primer (100 uM) | 2.5 | 32.5 |
dNTP (1 mM) | 0.8 | 10.4 |
10X Buffer | 2 | 26 |
Phi29 | 1 | 13 |
nfH2O | 8.7 | 113.1 |
Total | 20 | 195 |
Sample | Condition | Fraction | Lane | AmpR Index | Sample Vol (uL) | 2X Kapa SYBR qPCR MM | 10 uM Forward Primer | 10 uM Reverse Primer | H2O | Total Volume (uL) |
Sample 1A | Sample | Supernatant | A1 | 20 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1B | Sample | Supernatant | A2 | 20 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2A | No Template | Supernatant | A3 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2B | No Template | Supernatant | A4 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3A | No C Probe | Supernatant | A5 | 22 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3B | No C Probe | Supernatant | A6 | 22 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1A | Sample | Beads | A1 | 20 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1B | Sample | Beads | A2 | 20 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2A | No Template | Beads | A3 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2B | No Template | Beads | A4 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3A | No C Probe | Beads | A5 | 22 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3B | No C Probe | Beads | A6 | 22 | 2 | 25 | 1 | 1 | 21 | 50 |
NTC | qPCR Primers Only | A7 | 23 | 2 | 25 | 1 | 1 | 23 | 50 |
Results[edit]
Adapter Testing[edit]
Just like yesterday I'm going to test presence of C probes and circular product in the pre-RCA mix.
Master Mix | Primers | Primer Target | 2X Kapa SYBR qPCR MM | 10 uM Forward Primer | 10 uM Reverse Primer | H2O | Master Mix |
MMC1 | p24/p4* | C1 | 230 | 9.2 | 9.2 | 193.2 | MMC1 |
MMC2 | p24/p4* | C1 | 230 | 9.2 | 9.2 | 193.2 | MMC1 |
MMCP | p6/p12* | C2 | 230 | 9.2 | 9.2 | 193.2 | MMC2 |
Results[edit]
qPCR Sample Matrix[edit]
Sample | Condition | Primers | Primer Target | Fraction | Lane | Sample Vol (uL) | 2X Kapa SYBR qPCR MM | 10 uM Forward Primer | 10 uM Reverse Primer | H2O | Total Volume (uL) | Master Mix |
Sample 1A | Sample | p24/p4* | C1 | Supernatant-PreRCA | A1 | 2 | 25 | 1 | 1 | 21 | 50 | MMC1 |
Sample 1B | Sample | p24/p4* | C1 | Supernatant-PreRCA | A2 | 2 | 25 | 1 | 1 | 21 | 50 | MMC1 |
Sample 1A | Sample | p6/p12* | C2 | Supernatant-PreRCA | A3 | 2 | 25 | 1 | 1 | 21 | 50 | MMC2 |
Sample 1B | Sample | p6/p12* | C2 | Supernatant-PreRCA | A4 | 2 | 25 | 1 | 1 | 21 | 50 | MMC2 |
Sample 1A | Sample | p24/p12* | Circular Product | Supernatant-PreRCA | A5 | 2 | 25 | 1 | 1 | 21 | 50 | MMCP |
Sample 1B | Sample | p24/p12* | Circular Product | Supernatant-PreRCA | A6 | 2 | 25 | 1 | 1 | 21 | 50 | MMCP |
Sample 1A | Sample | p24/p4* | C1 | Beads-PreRCA | C1 | 2 | 25 | 1 | 1 | 21 | 50 | MMC1 |
Sample 1B | Sample | p24/p4* | C1 | Beads-PreRCA | C2 | 2 | 25 | 1 | 1 | 21 | 50 | MMC1 |
Sample 1A | Sample | p6/p12* | C2 | Beads-PreRCA | C3 | 2 | 25 | 1 | 1 | 21 | 50 | MMC2 |
Sample 1B | Sample | p6/p12* | C2 | Beads-PreRCA | C4 | 2 | 25 | 1 | 1 | 21 | 50 | MMC2 |
Sample 1A | Sample | p24/p12* | Circular Product | Beads-PreRCA | C5 | 2 | 25 | 1 | 1 | 21 | 50 | MMCP |
Sample 1B | Sample | p24/p12* | Circular Product | Beads-PreRCA | C6 | 2 | 25 | 1 | 1 | 21 | 50 | MMCP |
Sample 3A | No C Probes | p24/p4* | C1 | Supernatant-PreRCA | F1 | 2 | 25 | 1 | 1 | 21 | 50 | MMC1 |
Sample 3B | No C Probes | p24/p4* | C1 | Supernatant-PreRCA | F2 | 2 | 25 | 1 | 1 | 21 | 50 | MMC1 |
Sample 3A | No C Probes | p6/p12* | C2 | Supernatant-PreRCA | F3 | 2 | 25 | 1 | 1 | 21 | 50 | MMC2 |
Sample 3B | No C Probes | p6/p12* | C2 | Supernatant-PreRCA | F4 | 2 | 25 | 1 | 1 | 21 | 50 | MMC2 |
Sample 3A | No C Probes | p24/p12* | Circular Product | Supernatant-PreRCA | F5 | 2 | 25 | 1 | 1 | 21 | 50 | MMCP |
Sample 3B | No C Probes | p24/p12* | Circular Product | Supernatant-PreRCA | F6 | 2 | 25 | 1 | 1 | 21 | 50 | MMCP |
Sample 3A | No C Probes | p24/p4* | C1 | Beads-PreRCA | H1 | 2 | 25 | 1 | 1 | 21 | 50 | MMC1 |
Sample 3B | No C Probes | p24/p4* | C1 | Beads-PreRCA | H2 | 2 | 25 | 1 | 1 | 21 | 50 | MMC1 |
Sample 3A | No C Probes | p6/p12* | C2 | Beads-PreRCA | H3 | 2 | 25 | 1 | 1 | 21 | 50 | MMC2 |
Sample 3B | No C Probes | p6/p12* | C2 | Beads-PreRCA | H4 | 2 | 25 | 1 | 1 | 21 | 50 | MMC2 |
Sample 3A | No C Probes | p24/p12* | Circular Product | Beads-PreRCA | H5 | 2 | 25 | 1 | 1 | 21 | 50 | MMCP |
Sample 3B | No C Probes | p24/p12* | Circular Product | Beads-PreRCA | H6 | 2 | 25 | 1 | 1 | 21 | 50 | MMCP |
NTC | qPCR Primers Only | p24/p4* | C1 | NA | A7 | 2 | 25 | 1 | 1 | 23 | 50 | MMC1 |
NTC | qPCR Primers Only | p6/p12* | C2 | NA | C7 | 2 | 25 | 1 | 1 | 23 | 50 | MMC2 |
NTC | qPCR Primers Only | p24/p12* | Circular Product | NA | F7 | 2 | 25 | 1 | 1 | 23 | 50 | MMCP |