Daniel:Notebook/ComboLock/2017-2-3: Difference between revisions
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File:2017-02-03-v2Oligos-Round2-PostRCA.png|Gel Image-PostRCA | |||
File:|Gel Image-Epicentre | |||
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[[Category:ComboLock]] [[Category:20170202]] | [[Category:ComboLock]] [[Category:20170202]] |
Revision as of 23:34, 3 February 2017
Version 2 Oligos Round 2 (Started yesterday)
Protocol-Part 4-RCA
- Exonuclease Digestion
- Mix 15 uL Exo I (20U/uL) and 3 uL ExoIII (100U/uL)
- Add 2 uL to each sample, mix by swirling pipette tip
- Incubate for 1 hour at 37C
- Heat kill by incubating for 5 min at 95C
- Rolling Circle Amplification
- Prepare 8.2X Master mix according to table below; Give A samples LLRC and B Samples LLRC*
- Add 11.5 uL master mix to each tube
- Add sample and 1 uL appropriate enzyme to new reaction tubes
- Incubate at 37C for 3 hours
- Incubate at 65C for 10 minutes;Hold at 10C until next step
Reagent | uL Added | Master Mix (8.2X) | Master Mix (4.2X) |
Template | 5 | NA | NA |
RCA Primer (10 uM) | 2.5 | 0 | 0 |
dNTP (1 mM) | 5 | 41 | 21 |
10X Buffer | 2 | 16.4 | 8.4 |
Phi29 | 1 | 0 | 0 |
BSA (10 mg/mL) | 0.4 | 3.28 | 0 |
nfH2O | 4.1 | 33.62 | 18.9 |
Total | 20 | 114.8 | 58.8 |
Sample | 1X (AB) | 2X (AB) | 3X (AB) | 4X (AB) | 1Y (AB) | 4Y (AB) |
Phi29 Source | NEB | NEB | NEB | NEB | Epicentre | Epicentre |
Protocol Part 5-qPCR and Gel
- qPCR
- Make 21.2X qPCR master mix according to following recipe
- 445.2 uL nfH2O
- 530 uL SYBR Fast
- 21.2 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 224 uL TBE, 56 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 |
Sample 1AY | Sample | Post | D1 | 28 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1BY | Sample | Post | D2 | 28 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4AY | No Lock Phosphate | Post | D3 | 29 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4BY | No Lock Phosphate | Post | D4 | 29 | 2 | 25 | 1 | 1 | 21 | 50 |
NTC | qPCR Primers Only | D5 | 30 | 2 | 25 | 1 | 1 | 23 | 50 |
Results
- 2017-02-03-v2Oligos-Round2-PostRCA.png
Gel Image-PostRCA