Daniel:Notebook/ComboLock/2017-1-19: Difference between revisions
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===Results=== | ===Results=== | ||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#92CDDC;font-size:12pt;font-weight:bold" align="center" | |||
| width="91" height="24" | Sample Name | |||
| width="60" | 1A | |||
| width="76" | 1B | |||
| width="71" | 1AY | |||
| width="65" | 1BY | |||
| width="65" | 1AF | |||
| width="65" | 1BF | |||
| width="65" | 4AF | |||
| width="65" | 4BF | |||
| width="65" | 1AR | |||
| width="65" | 1BR | |||
| width="65" | 4AR | |||
| width="65" | 4BR | |||
|- style="font-size:12pt" | |||
| height="45" | Sample Condition | |||
| align="center" | Sample-PreRCA | |||
| align="center" | Sample-PreRCA | |||
| align="center" | Sample | |||
| align="center" | Sample | |||
| align="center" | Sample | |||
| align="center" | Sample | |||
| align="center" | No Phosphate (PCCB) | |||
| align="center" | No Phosphate (PCCB) | |||
| align="center" | Sample | |||
| align="center" | Sample | |||
| align="center" | No Phosphate (PCCB) | |||
| align="center" | No Phosphate (PCCB) | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="30" | RCA Primer | |||
| align="center" | None | |||
| align="center" | None | |||
| align="center" | LLRC (Mine) | |||
| align="center" | LLRC (Mine) | |||
| align="center" | AmpF | |||
| align="center" | AmpF | |||
| align="center" | AmpF | |||
| align="center" | AmpF | |||
| align="center" | AmpR | |||
| align="center" | AmpR | |||
| align="center" | AmpR | |||
| align="center" | AmpR | |||
|- style="font-size:12pt" | |||
| height="15" | Conc (ng/uL) | |||
| align="center" align="center" | 0.81 | |||
| align="center" align="center" | 1.27 | |||
| align="center" align="center" | 0.72 | |||
| align="center" align="center" | 0.62 | |||
| align="center" align="center" | 35.1 | |||
| align="center" align="center" | 46.9 | |||
| align="center" align="center" | 35 | |||
| align="center" align="center" | 40.7 | |||
| align="center" align="center" | 25.3 | |||
| align="center" align="center" | 26.8 | |||
| align="center" align="center" | 31.5 | |||
| align="center" align="center" | 30.2 | |||
|} | |||
[[Category:ComboLock]] [[Category:20170113]] | [[Category:ComboLock]] [[Category:20170113]] |
Revision as of 18:34, 20 January 2017
Version 3 Oligos (Started Thursday, Jan 13)
RCA Repeat (Started Yesterday)
- qPCR
- Make qPCR master mix according to following table
- Add 47 (MM1) or 48 (MM2) uL master mix to each well
- Add 2 uL sample and 1 uL appropriate Reverse Primer (10 uM, MM1 only) according to table
- 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 80 uL TBE, 20 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 | Primer Pair | Forward Primer (10 uM) uL | Reverse Primer (10 uM) uL | uL nfH2O | uL Kapa SYBR Fast Master Mix 2X |
MM1 | AmpF/AmpR | 9.2 | NA | 193.2 | 230 |
MM2 | Primer12RC/AmpRInd25 | 9.2 | 9.2 | 193.2 | 230 |
Sample | Condition | Lane | Primer Pair | AmpR Index | 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 | A1 | AmpF/AmpR | 21 | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 1B | Sample | A2 | AmpF/AmpR | 21 | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 2A | No Template | A3 | AmpF/AmpR | 22 | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 2B | No Template | A4 | AmpF/AmpR | 22 | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 3A | No C Probe | A5 | AmpF/AmpR | 23 | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 3B | No C Probe | A6 | AmpF/AmpR | 23 | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 4A | No PCCB Phosphate | A7 | AmpF/AmpR | 24 | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 4B | No PCCB Phosphate | A8 | AmpF/AmpR | 24 | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 1A | Sample-PostRCA | H1 | Primer12RC/AmpR | 25 | 2 | 25 | 1 | 1 | 21 | 50 | MM2 |
Sample 1B | Sample-PostRCA | H2 | Primer12RC/AmpR | 25 | 2 | 25 | 1 | 1 | 21 | 50 | MM2 |
Sample 4A | No PCCB Phosphate | H3 | Primer12RC/AmpR | 25 | 2 | 25 | 1 | 1 | 21 | 50 | MM2 |
Sample 4B | No PCCB Phosphate | H4 | Primer12RC/AmpR | 25 | 2 | 25 | 1 | 1 | 21 | 50 | MM2 |
Sample 1A | Sample-PreRCA | H5 | Primer12RC/AmpR | 25 | 2 | 25 | 1 | 1 | 21 | 50 | MM2 |
Sample 1B | Sample-PreRCA | H6 | Primer12RC/AmpR | 25 | 2 | 25 | 1 | 1 | 21 | 50 | MM2 |
Sample 4A | No PCCB PO4-PreRCA | H7 | Primer12RC/AmpR | 25 | 2 | 25 | 1 | 1 | 21 | 50 | MM2 |
Sample 4B | No PCCB PO4-PreRCA | H8 | Primer12RC/AmpR | 25 | 2 | 25 | 1 | 1 | 21 | 50 | MM2 |
NTC | qPCR Primers Only | D1 | AmpF/AmpR | 26 | 2 | 25 | 1 | 1 | 23 | 50 | MM1 |
NTC | qPCR Primers Only | D2 | Primer12RC/AmpR | 25 | 2 | 25 | 1 | 1 | 23 | 50 | MM2 |
Results
- 20170119-qPCR-v3Oligos-AmpFAmpR.png
qPCR Curve-AmpF and AmpR
- 20170119-qPCR-v3Oligos-Primer12RCAmpR.png
qPCR Curve-Primer12RC and AmpR
- 2017-01-19-v3Oligos-RCA-2.png
Gel Image-AmpF and AmpR
- 2017-01-19-v3Oligos-NoAmpF.png
Gel Image-Primer12RC and AmpR
Discussion
Ummmm. So the AmpR/Primer12RC should only amplify circular product (yesterday's p12/p24 combo is good for circular product and bridge). So once again it showed up in both pre and post RCA reactions, but the normal AmpR/AmpF pairing (necessary for sequencing) showed nothing. Also, assuming I didn't mix up the samples, it kinda seems like there is more product Pre-RCA than post. Going off the qPCR curves (generally a bad idea but probably comparable between these two) it looks like the "PreRCA" sample fraction is ~4X more than the "PostRCA" fraction. This actually makes a ton of sense if there was no amplification from RCA. Both PCRs had 2 uL sample added to them, but the "PostRCA" fraction got 5 uL (out of 20) of "PreRCA" fraction as input. So basically I put the "PreRCA" fraction into water, or might as well have.
RCA Test
Since it seems like its RCA that is the problem I will do RCA again, this time testing both AmpF and AmpR as (dirty) primers. To save on sample space, I'll use 1 and 4 again (4 is a suitable negative control). I should know whether or not the product amplifies in even half an hour, so we're gonna go with that (measure using Qubit)
- Rolling Circle Amplification
- Prepare 8.5X Master mix according to table below
- Add 15 uL master mix to each tube
- Add sample to new reaction tubes according to following table
- Incubate at 37C for 30 minutes
- Incubate at 65C for 10 minutes;Hold at 10C until next step
- Qubit Measure
- Mix dye to Qubit buffer; 10 uL dye in 1990 uL buffer (1:200 ratio per sample)
- Add 190 uL dye/buffer and 10 uL standard to standard tubes
- Add 199 uL dye/buffer and 1 uL sample to sample tubes
- Measure in Qubit
Reagent | uL Added | Master Mix (8.5X) |
Template | 5 | NA |
RCA Primer (AmpF or AmpRInd21; 10 uM) | 2.5 | 21.25 |
dNTP (1 mM) | 0.8 | 6.8 |
10X Phi29 Buffer | 2 | 17 |
Phi29 | 1 | 8.5 |
nfH2O | 8.7 | 73.95 |
Total | 20 | 127.5 |
Results
Sample Name | 1A | 1B | 1AY | 1BY | 1AF | 1BF | 4AF | 4BF | 1AR | 1BR | 4AR | 4BR |
Sample Condition | Sample-PreRCA | Sample-PreRCA | Sample | Sample | Sample | Sample | No Phosphate (PCCB) | No Phosphate (PCCB) | Sample | Sample | No Phosphate (PCCB) | No Phosphate (PCCB) |
RCA Primer | None | None | LLRC (Mine) | LLRC (Mine) | AmpF | AmpF | AmpF | AmpF | AmpR | AmpR | AmpR | AmpR |
Conc (ng/uL) | 0.81 | 1.27 | 0.72 | 0.62 | 35.1 | 46.9 | 35 | 40.7 | 25.3 | 26.8 | 31.5 | 30.2 |