Daniel:Notebook/ComboLock/2017-1-24
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Circularization Test (Started Yesterday)[edit]
Protocol-Part 2[edit]
- 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 1.5 hours at 37C
- Heat kill by incubating for 5 min at 95C
- Rolling Circle Amplification
- Prepare 7X 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 3 hours
- Incubate at 65C for 10 minutes;Hold at 10C until next step
- qPCR
- Make qPCR master mix according to following recipes
- Add 47 (MM1) or 48 (MM2) uL master mix to each well
- Add 2 uL sample and/or 1 uL appropriate Reverse Primer (1 uM) according to table in 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 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
Reagent | uL Added | Master Mix (8.5X) |
Template | 5 | NA |
RCA Primer (10 uM) | 2.5 | 21.25 |
dNTP (1 mM) | 0.8 | 6.8 |
10X Buffer | 2 | 17 |
Phi29 | 1 | 8.5 |
nfH2O | 8.7 | 73.95 |
Total | 20 | 127.5 |
Protocol-Part 3[edit]
Master Mix | Primer Pair | uL Forward Primer | uL Reverse Primer | uL nfH2O | uL 2X SYBR Fast MM | uL Total |
MM1 | AmpF/AmpR | 17.2 | None | 361.2 | 430 | 808.4 |
MM2 | primer6RC/AmpR.Ind29 | 9.2 | 9.2 | 193.2 | 230 | 441.6 |
qPCR Sample Table[edit]
Sample | Product Amt | Pre/Post RCA | Primer Pair | Lane | 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 0A | 1pmol | Pre | AmpF/AmpR | A1 | 21 | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 0B | 1pmol | Pre | AmpF/AmpR | A2 | 21 | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 2A | 10 fmol | Pre | AmpF/AmpR | A3 | 22 | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 2B | 10 fmol | Pre | AmpF/AmpR | A4 | 22 | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 4A | 100 amol | Pre | AmpF/AmpR | A5 | 23 | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 4B | 100 amol | Pre | AmpF/AmpR | A6 | 23 | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 6A | 1 amol | Pre | AmpF/AmpR | A7 | 24 | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 6B | 1 amol | Pre | AmpF/AmpR | A8 | 24 | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 0AX | 1pmol | Post | AmpF/AmpR | H1 | 25 | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 0BX | 1pmol | Post | AmpF/AmpR | H2 | 25 | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 2AX | 10 fmol | Post | AmpF/AmpR | H3 | 26 | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 2BX | 10 fmol | Post | AmpF/AmpR | H4 | 26 | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 4AX | 100 amol | Post | AmpF/AmpR | H5 | 27 | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 4BX | 100 amol | Post | AmpF/AmpR | H6 | 27 | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 6AX | 1 amol | Post | AmpF/AmpR | H7 | 28 | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 6BX | 1 amol | Post | AmpF/AmpR | H8 | 28 | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 0A | 1pmol | Pre | primer6RC/AmpR | C1 | 29 | 2 | 25 | 1 | 1 | 21 | 50 | MM2 |
Sample 0B | 1pmol | Pre | primer6RC/AmpR | C2 | 29 | 2 | 25 | 1 | 1 | 21 | 50 | MM2 |
Sample 6A | 1 amol | Pre | primer6RC/AmpR | C3 | 29 | 2 | 25 | 1 | 1 | 21 | 50 | MM2 |
Sample 6B | 1 amol | Pre | primer6RC/AmpR | C4 | 29 | 2 | 25 | 1 | 1 | 21 | 50 | MM2 |
Sample 0AX | 1pmol | Post | primer6RC/AmpR | C5 | 29 | 2 | 25 | 1 | 1 | 21 | 50 | MM2 |
Sample 0BX | 1pmol | Post | primer6RC/AmpR | C6 | 29 | 2 | 25 | 1 | 1 | 21 | 50 | MM2 |
Sample 6AX | 1 amol | Post | primer6RC/AmpR | C7 | 29 | 2 | 25 | 1 | 1 | 21 | 50 | MM2 |
Sample 6BX | 1 amol | Post | primer6RC/AmpR | C8 | 29 | 2 | 25 | 1 | 1 | 21 | 50 | MM2 |
NTC | qPCR Primers Only | NA | AmpF/AmpR | F1 | 30 | 2 | 25 | 1 | 1 | 23 | 50 | MM1 |
NTC | qPCR Primers Only | NA | primer6RC/AmpR | F2 | 30 | 2 | 25 | 1 | 1 | 23 | 50 | MM2 |
Results-Curves and Gel Images[edit]
- 20170124-qPCR-PrePostRCA.png
qPCR Curve
- 2017-01-24-RCATest-PreRCA.png
Gel image-Pre RCA
- 2017-01-24-RCATest-PostRCA.png
Gel image-Post RCA
- 2017-01-24-RCATest-Adapters.png
Gel image-Adapters
- 20170124-qPCR-SeqVsAdap-RCA.png
qPCR Curve
Results-Cycle Threshold[edit]
Sample | Lanes | CT Avg | CT Diff to PreRCA | Amplification | CT Diff to 1 amol | Amplification to 1 amol |
1 pmol PreRCA | A1,A2 | 17.6 | NA | NA | 1.2 | 2.3 |
10 fmol PreRCA | A3,A4 | 19.1 | NA | NA | -0.1 | 0.8 |
100 amol PreRCA | A5,A6 | 18.9 | NA | NA | -0.2 | 1.0 |
1 amol PreRCA | A7,A8 | 18.9 | NA | NA | 0.0 | 1.0 |
1 pmol PostRCA | H1,H2 | 11.4 | 6.25 | 75.8 | 6.9 | 119.8 |
10 fmol PostRCA | H3,H4 | 16.3 | 2.78 | 6.8 | 2.0 | 3.9 |
100 amol PostRCA | H5,H6 | 18.2 | 0.69 | 1.6 | 0.1 | 1.1 |
1 amol PostRCA | H7,H8 | 18.3 | 0.56 | 1.5 | 0.0 | 1.0 |
1 pmol PreRCA Adapters | C1,C2 | 16.1 | NA | NA | 5.1 | 33.4 |
1 amol PreRCA Adapters | C3,C4 | 21.1 | NA | NA | 0.0 | 1.0 |
1 pmol PostRCA Adapters | C5,C6 | 11.3 | 4.8 | 27.5 | 9.8 | 916.5 |
1 amol PostRCA Adapters | C7,C8 | 20.8 | 0.4 | 1.3 | 0.4 | 1.3 |