Daniel:Notebook/ComboLock/2016-12-15: Difference between revisions
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>Djacobse |
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I'm now going to investigate the presence/absence of C probes and circularized products using the adapter primers. | I'm now going to investigate the presence/absence of C probes and circularized products using the adapter primers. | ||
<ol start="7"> | |||
<li>qPCR</li> | |||
<ol type="A"> | |||
<li>Make master mix according to following recipes</li> | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#B1A0C7;font-size:12pt;font-weight:bold" align="center" | |||
| width="85" height="45" | Master Mix | |||
| width="85" | Primers | |||
| width="65" | Primer Target | |||
| width="85" | 2X Kapa SYBR qPCR MM | |||
| width="85" | 10 uM Forward Primer | |||
| width="85" | 10 uM Reverse Primer | |||
| width="65" | H2O | |||
| width="65" | Master Mix | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" | MMC1 | |||
| align="center" | p24/p4* | |||
| align="center" | C1 | |||
| align="center" align="center" | 230 | |||
| align="center" align="center" | 9.2 | |||
| align="center" align="center" | 9.2 | |||
| align="center" align="center" | 193.2 | |||
| align="center" | MMC1 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
|style="font-weight:bold" height="15" | MMC2 | |||
| align="center" | p24/p4* | |||
| align="center" | C1 | |||
| align="center" align="center" | 230 | |||
| align="center" align="center" | 9.2 | |||
| align="center" align="center" | 9.2 | |||
| align="center" align="center" | 193.2 | |||
| align="center" | MMC1 | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" | MMCP | |||
| align="center" | p6/p12* | |||
| align="center" | C2 | |||
| align="center" align="center" | 230 | |||
| align="center" align="center" | 9.2 | |||
| align="center" align="center" | 9.2 | |||
| align="center" align="center" | 193.2 | |||
| align="center" | MMC2 | |||
|} | |||
<li>Add 47 uL master mix to each well</li> | |||
<li>Add 2 uL sample and 1 uL appropriate Reverse Primer (1 uM) according to table in the following section</li> | |||
===qPCR Sample Matrix=== | ===qPCR Sample Matrix=== |
Revision as of 19:15, 15 December 2016
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 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
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 |
qPCR Sample Matrix
Sample | Condition | Primers | Primer Target | 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 | p24/p4* | C1 | Supernatant-PreRCA | A1 | 20 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1B | Sample | p24/p4* | C1 | Supernatant-PreRCA | A2 | 20 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1A | Sample | p6/p12* | C2 | Supernatant-PreRCA | A3 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1B | Sample | p6/p12* | C2 | Supernatant-PreRCA | A4 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1A | Sample | p24/p12* | Circular Product | Supernatant-PreRCA | A3 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1B | Sample | p24/p12* | Circular Product | Supernatant-PreRCA | A4 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1A | Sample | p24/p4* | C1 | Beads-PreRCA | A1 | 20 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1B | Sample | p24/p4* | C1 | Beads-PreRCA | A2 | 20 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1A | Sample | p6/p12* | C2 | Beads-PreRCA | A3 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1B | Sample | p6/p12* | C2 | Beads-PreRCA | A4 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1A | Sample | p24/p12* | Circular Product | Beads-PreRCA | A3 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1B | Sample | p24/p12* | Circular Product | Beads-PreRCA | A4 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3A | No C Probes | p24/p4* | C1 | Supernatant-PreRCA | A1 | 20 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3B | No C Probes | p24/p4* | C1 | Supernatant-PreRCA | A2 | 20 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3A | No C Probes | p6/p12* | C2 | Supernatant-PreRCA | A3 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3B | No C Probes | p6/p12* | C2 | Supernatant-PreRCA | A4 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3A | No C Probes | p24/p12* | Circular Product | Supernatant-PreRCA | A3 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3B | No C Probes | p24/p12* | Circular Product | Supernatant-PreRCA | A4 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3A | No C Probes | p24/p4* | C1 | Beads-PreRCA | A1 | 20 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3B | No C Probes | p24/p4* | C1 | Beads-PreRCA | A2 | 20 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3A | No C Probes | p6/p12* | C2 | Beads-PreRCA | A3 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3B | No C Probes | p6/p12* | C2 | Beads-PreRCA | A4 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3A | No C Probes | p24/p12* | Circular Product | Beads-PreRCA | A3 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3B | No C Probes | p24/p12* | Circular Product | Beads-PreRCA | A4 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
NTC | qPCR Primers Only | p24/p4* | C1 | NA | A7 | 26 | 2 | 25 | 1 | 1 | 23 | 50 |
NTC | qPCR Primers Only | p6/p12* | C2 | NA | A7 | 26 | 2 | 25 | 1 | 1 | 23 | 50 |
NTC | qPCR Primers Only | p24/p12* | Circular Product | NA | A7 | 26 | 2 | 25 | 1 | 1 | 23 | 50 |