Daniel:Notebook/ComboLock/2017-1-19: Difference between revisions
>Djacobse |
>Djacobse |
||
Line 473: | Line 473: | ||
|} | |} | ||
===Discussion (RCA Test)=== | |||
From the results on the table, I'd say there was amplification (on the order of 35X for sample 1) with AmpF '''and''' AmpR. Based on the potential dirtiness of the AmpF and AmpR primers, I'm going to repeat the experiment tomorrow using my adapter primers. | |||
Continued [[Daniel:Notebook/ComboLock/2017-1-20|tomorrow]] | |||
[[Category:ComboLock]] [[Category:20170113]] | [[Category:ComboLock]] [[Category:20170113]] |
Latest revision as of 18:37, 20 January 2017
Version 3 Oligos (Started Thursday, Jan 13)[edit]
RCA Repeat (Started Yesterday)[edit]
- 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[edit]
- 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[edit]
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[edit]
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[edit]
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 |
Discussion (RCA Test)[edit]
From the results on the table, I'd say there was amplification (on the order of 35X for sample 1) with AmpF and AmpR. Based on the potential dirtiness of the AmpF and AmpR primers, I'm going to repeat the experiment tomorrow using my adapter primers.
Continued tomorrow