Daniel:Notebook/ComboLock/2017-2-8: Difference between revisions
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=v3 Oligos-Round 3= | =v3 Oligos-Round 3-Low Temp (Started [[Daniel:Notebook/ComboLock/2017-2-7|Yesterday]])= | ||
[[Daniel:Notebook/ComboLock|Back to Calendar]] | [[Daniel:Notebook/ComboLock|Back to Calendar]] | ||
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<li>Rinse gel and image in gel doc</li> | <li>Rinse gel and image in gel doc</li> | ||
</ol></ol> | </ol></ol> | ||
==Results-Ethanol Precipitation== | ==Results-Ethanol Precipitation== |
Latest revision as of 19:14, 16 February 2017
v3 Oligos-Round 3-Low Temp (Started Yesterday)[edit]
Protocol-Part 4[edit]
- qPCR
- Make 17.2X qPCR master mix according to following recipe
- 361.2 uL nfH2O
- 430 uL SYBR Fast
- 17.2 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
- 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 192 uL TBE, 48 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 23 minutes at 230V
- Open gel and stain with 3 uL SYBR Gold for 3 minutes
- Rinse gel and image in gel doc
Sample | Condition | Pre/Post RCA | 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 | Pre | A1 | 20 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1B | Sample | Pre | A2 | 20 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2A | No Template | Pre | A3 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2B | No Template | Pre | A4 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3A | No C Probe | Pre | A5 | 22 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3B | No C Probe | Pre | A6 | 22 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4A | No Lock Phosphate | Pre | A7 | 23 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4B | No Lock Phosphate | Pre | A8 | 23 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1AX | Sample | Post | H1 | 24 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1BX | Sample | Post | H2 | 24 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2AX | No Template | Post | H3 | 25 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2BX | No Template | Post | H4 | 25 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3AX | No C Probe | Post | H5 | 26 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3BX | No C Probe | Post | H6 | 26 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4AX | No Lock Phosphate | Post | H7 | 27 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4BX | No Lock Phosphate | Post | H8 | 27 | 2 | 25 | 1 | 1 | 21 | 50 |
NTC | qPCR Primers Only | D5 | 30 | 2 | 25 | 1 | 1 | 23 | 50 |
Results-Normal Protocol[edit]
- 20170208-qPCR-v3Oligos-LoTemp.png
qPCR curve-Normal protocol
- 2017-02-08-v3Oligos-LoTemp-PreRCA.png
Gel image-Pre RCA
- 2017-02-08-v3Oligos-LoTemp-PostRCA.png
Gel image-Post RCA
- 2017-02-08-v3Oligos-LoTemp-PostRCA-Highlight.png
Gel image-Post RCA-Product Highlighted
As the highlighted band shows, sample A might actually have amplified since there is a faint band at the correct size that is not in the other lanes. However, it is faint and about equal to the incorrect band at 200bp.
Protocol-Part 5[edit]
- Ethanol Precipitation
- Centrifuge at 4C for 30 minutes at 12000 rpm
- Remove supernatant and add 750 uL cold 70%EtOH
- Centrifuge at 4C for 20 minutes at 12000 rpm
- Remove supernatant and vacuum centrifuge for 15 minutes to completely dry
- Resuspend pellet in 20 uL nfH2O
- Rolling Circle Amplification
- Prepare 8.2X Master mix according to table below; use LLRC primer for A samples and LLRC* (phosphorothiorate) samples for B samples
- Add 15 uL master mix to each tube
- Add sample to new reaction tubes according to following table
- Incubate at 30C for 6 hours
- Incubate at 65C for 10 minutes;Hold at 10C until next step
Reagent | uL Added | Master Mix (8.2X) |
Template | 3 | 0 |
RCA Primer (10 uM) | 2.5 | 20.5 |
dNTP (1 mM) | 0.8 | 6.56 |
10X Buffer | 2 | 16.4 |
Phi29 | 1 | 0 |
nfH2O | 10.7 | 87.74 |
Total | 20 | 131.2 |
Protocol-Part 6[edit]
- qPCR
- Make 17.2X qPCR master mix according to following recipe
- 361.2 uL nfH2O
- 430 uL SYBR Fast
- 17.2 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
- 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 192 uL TBE, 48 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 23 minutes at 230V
- Open gel and stain with 3 uL SYBR Gold for 3 minutes
- Rinse gel and image in gel doc
- TBE Gel-Unamplified EtOH Precipitation product
- Mix 60 uL TBE, 20 uL 6x loading dye
- Aliquot 8 uL per sample/ladder lane onto parafilm
- Add 4 uL of sample or 1 uL ladder, 3 uL TBE to correct drop
- Load 10 uL in to well
- Run gel for 23 minutes at 230V
- Open gel and stain with 2 uL SYBR Gold for 3 minutes
- Rinse gel and image in gel doc
Sample | Condition | Pre/Post RCA | 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-EtOH+ | Pre | A1 | 20 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1B* | Sample-EtOH+ | Pre | A2 | 20 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2A* | No Template-EtOH+ | Pre | A3 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2B* | No Template-EtOH+ | Pre | A4 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3A* | No C Probe-EtOH+ | Pre | A5 | 22 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3B* | No C Probe-EtOH+ | Pre | A6 | 22 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4A* | No Lock Phosphate-EtOH+ | Pre | A7 | 23 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4B* | No Lock Phosphate-EtOH+ | Pre | A8 | 23 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1AZ | Sample-EtOH+ | Post | H1 | 24 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1BZ | Sample-EtOH+ | Post | H2 | 24 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2AZ | No Template-EtOH+ | Post | H3 | 25 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2BZ | No Template-EtOH+ | Post | H4 | 25 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3AZ | No C Probe-EtOH+ | Post | H5 | 26 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3BZ | No C Probe-EtOH+ | Post | H6 | 26 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4AZ | No Lock Phosphate-EtOH+ | Post | H7 | 27 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4BZ | No Lock Phosphate-EtOH+ | Post | H8 | 27 | 2 | 25 | 1 | 1 | 21 | 50 |
NTC | qPCR Primers Only | D5 | 30 | 2 | 25 | 1 | 1 | 23 | 50 |
Results-Ethanol Precipitation[edit]
- 20170208-qPCR-v3Oligos-LoTemp-EtOH.png
qPCR curve-Normal protocol
- 2017-02-08-v3Oligos-LoTemp-PreRCA-EtOH.png
Gel image-Pre RCA
- 2017-02-08-v3Oligos-LoTemp-PostRCA-EtOH.png
Gel image-Post RCA
- 2017-02-08-v3Oligos-LoTemp-AfterEtOH.png
Purified samples after EtOH before amplification