Daniel:Notebook/ComboLock/2016-12-15
Mirror Probe Test (Started Tuesday)
RCA on Beads/Supernatant Fractions
- qPCR
- Make master mix according to following recipe
- 298.2 uL nfH2O
- 355 uL SYBR Fast
- 14.2 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 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
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 | H1 | 23 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1B | Sample | Beads | H2 | 23 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2A | No Template | Beads | H3 | 24 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2B | No Template | Beads | H4 | 24 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3A | No C Probe | Beads | H5 | 25 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3B | No C Probe | Beads | H6 | 25 | 2 | 25 | 1 | 1 | 21 | 50 |
NTC | qPCR Primers Only | NA | A7 | 26 | 2 | 25 | 1 | 1 | 23 | 50 |
Results
- 20161215-qPCR-MirrorCProbe-PostRCA.png
qPCR curves
- 2016-12-15-MirrorCProbe-RCA-Supernatant.png
Gel image-supernatant RCA
- 2016-12-15-MirrorCProbe-RCA-Beads.png
Gel image-beads RCA
Although there is a slight smear in the region of correct size (211bp), the smear is present in every lane (except the NTC). This suggests the smear is a result of the beads in general and not really related to the product. My conclusion is that the circular product never formed.
C Probe and Product Detection
I'm now going to investigate the presence/absence of C probes and circularized products using the adapter primers.
- 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
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
- 20161215-qPCR-MirrorCProbes-Adapters-SamplevsControl-Beads.png
qPCR curve displaying sample vs C probes (for supernatant)
- 20161215-qPCR-MirrorCProbes-Adapters-SamplevsControl.png
qPCR curve displaying sample vs C probes (for beads)
- 20161215-qPCR-MirrorCProbes-Adapters-SupvsBeads.png
qPCR curve displaying supernatant vs. beads effect
- 2016-12-15-MirrorCProbe-Adapters-CProbeDetection.png
Gel image 1-Sample C probe detection
- 2016-12-15-MirrorCProbe-Adapters-Controls.png
Gel image 2-Controls gel
qPCR Sample Matrix
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 |
Overnight Capture
So based on the results above, I'd say that the latch and padlock are not both finding their targets at the same time, which is leading to a situation where nothing works. So I'm going to repeat the experiment but with an overnight capture step that will give everything time to settle. I'll incubate at 60C overnight for capture (The Tm of the adapters is designed to 65 btw). Tomorrow I will add the phusion reaction (Phusion polymerase + Amp Ligase) for 2 hours without taking the samples off the incubator.
Protocol
- Template-Bead Binding
- Suspend 2 uL (8 ug) beads per sample in 100 uL wash buffer
- Apply magnet for 30 sec and remove supernatant
- Add 2 uL 10 uM template oligo per sample to bead solution; incubate at RT for 5 min
- Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant
- C Probe Hybridization
- Combine 1 uL of each C probe (10 uM stock) and 3 uL bead wash buffer into a 0.2 mL tube
- Heat C probes to 90C for 5 min; Chill probes on ice to quench
- Add 5 uL probe mixture to beads
- Incubate at 40C for 1 hour with agitation
- Wash beads with 100 uL wash buffer; apply magnet and remove supernatant
- Repeat wash step above
- Add 100 uL cold Low Salt Buffer; apply magnet and remove supernatant
- Latch and Padlock Hybridization
- Add following to a fresh 0.2 mL tube
- 12.2 uL Latch0001, 10 uM stock
- 12.2 uL Padlock0201, 10 uM stock
- 12.2 uL Amp10X buffer
- 85.4 uL nfH2O
- Incubate at 60C overnight
Continued tomorrow