Daniel:Notebook/ComboLock/2017-1-25
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Circularization Test (Started Monday)
Protocol-New Amplification Primers
These are new primers I designed. They are slightly adjusted from the locations of AmpF/AmpR. Hopefully there is less noise.
- qPCR
- Make qPCR master mix according to following recipe
- 361.2 uL nfH2O
- 17.2 uL AmpF-CLv3
- 17.2 uL AmpR-CLv3
- 430 uL Kapa SYBR Fast
- Add 48 uL master mix to each well
- Add 2 uL sample 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
Sample | Product Amt | Pre/Post RCA | Lane | Sample Vol (uL) | 2X Kapa SYBR qPCR MM | 10 uM Forward Primer | 10 uM Reverse Primer | H2O | Total Volume (uL) |
Sample 0A | 1pmol | Pre | A1 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 0B | 1pmol | Pre | A2 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2A | 10 fmol | Pre | A3 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2B | 10 fmol | Pre | A4 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4A | 100 amol | Pre | A5 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4B | 100 amol | Pre | A6 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 6A | 1 amol | Pre | A7 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 6B | 1 amol | Pre | A8 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 0AX | 1pmol | Post | H1 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 0BX | 1pmol | Post | H2 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2AX | 10 fmol | Post | H3 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2BX | 10 fmol | Post | H4 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4AX | 100 amol | Post | H5 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4BX | 100 amol | Post | H6 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 6AX | 1 amol | Post | H7 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 6BX | 1 amol | Post | H8 | 2 | 25 | 1 | 1 | 21 | 50 |
Results
- 20170125-qPCR-NewAmp.png
qPCR Curve
- 2017-01-25-RCATest-NewAmp-Pre.png
Gel image-Pre RCA
- 2017-01-25-RCATest-NewAmp-Post.png
Gel image-Post RCA
RCA with Epicentre Phi29
Still have some left, so I'm going to test its effectiveness by trying to amplify the 1 amol sample. I'll use the original Epicentre concentration and a 10X dilution, since the Epicentre Phi29 is 10X more concentrated than NEB.
- 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
Reagent | uL Added | Master Mix (4.5X) |
Template | 5 | NA |
RCA Primer (LLRC 10 uM) | 2.5 | 11.25 |
dNTP (1 mM) | 0.8 | 3.6 |
10X Buffer | 2 | 9 |
Phi29 | 1 | 0 |
nfH2O | 8.7 | 39.15 |
Total | 20 | 63 |