Daniel:Notebook/ComboLock/2016-10-24
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Cell Test 3 (Started Friday 10/21)[edit]
I'm going to try some more qPCR runs in order to delve into this deeper. For one, I'm going to repeat the qPCR from Saturday for time points T1 and T2, since the results are a little strange (I saw nothing in T1 and T2). Second, I'm going to test the adapter based amplifications for latch padlock for several time points, including the last product.
qPCR Repeats[edit]
- qPCR
- Prepare the following master mixes according to table
- Add 48 uL master mix to each well
- Add 2 uL sample to correct well by following sample table in next 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 56 uL TBE, 14 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 | Forward/Reverse | Forward Primer (uL) | Reverse Primer (uL) | nfH2O (uL) | Kapa SYBR Fast MM (uL) |
MM1 | p24RC/p4 | 0.9 | 0.9 | 207.5 | 227.5 |
MM2 | p6RC/p12 | 0.9 | 0.9 | 207.5 | 227.5 |
qPCR Sample Table[edit]
Sample | Condition | Time Point | Lane | AmpR-F Combo | Sample Vol (uL) | 2X Kapa SYBR qPCR MM | 100 uM Forward Primer | 100 uM Reverse Primer | H2O | Total Volume (uL) | Master Mix |
Sample 1 | Exp. Rep 1 | T1 | A1 | p24RC/p4 | 2 | 25 | 0.1 | 0.1 | 22.8 | 50 | MM1 |
Sample 2 | Exp. Rep 2 | T1 | B1 | p24RC/p4 | 2 | 25 | 0.1 | 0.1 | 22.8 | 50 | MM1 |
Sample 3 | No C Probes | T1 | C1 | p24RC/p4 | 2 | 25 | 0.1 | 0.1 | 22.8 | 50 | MM1 |
Sample 4 | No Cells | T1 | D1 | p24RC/p4 | 2 | 25 | 0.1 | 0.1 | 22.8 | 50 | MM1 |
Sample 1 | Exp. Rep 1 | T1 | E1 | p6RC/p12 | 2 | 25 | 0.1 | 0.1 | 22.8 | 50 | MM2 |
Sample 2 | Exp. Rep 2 | T1 | F1 | p6RC/p12 | 2 | 25 | 0.1 | 0.1 | 22.8 | 50 | MM2 |
Sample 3 | No C Probes | T1 | G1 | p6RC/p12 | 2 | 25 | 0.1 | 0.1 | 22.8 | 50 | MM2 |
Sample 4 | No Cells | T1 | H1 | p6RC/p12 | 2 | 25 | 0.1 | 0.1 | 22.8 | 50 | MM2 |
Sample 1 | Exp. Rep 1 | T2 | A3 | p24RC/p4 | 2 | 25 | 0.1 | 0.1 | 22.8 | 50 | MM1 |
Sample 2 | Exp. Rep 2 | T2 | B3 | p24RC/p4 | 2 | 25 | 0.1 | 0.1 | 22.8 | 50 | MM1 |
Sample 3 | No C Probes | T2 | C3 | p24RC/p4 | 2 | 25 | 0.1 | 0.1 | 22.8 | 50 | MM1 |
Sample 4 | No Cells | T2 | D3 | p24RC/p4 | 2 | 25 | 0.1 | 0.1 | 22.8 | 50 | MM1 |
Sample 1 | Exp. Rep 1 | T2 | E3 | p6RC/p12 | 2 | 25 | 0.1 | 0.1 | 22.8 | 50 | MM2 |
Sample 2 | Exp. Rep 2 | T2 | F3 | p6RC/p12 | 2 | 25 | 0.1 | 0.1 | 22.8 | 50 | MM2 |
Sample 3 | No C Probes | T2 | G3 | p6RC/p12 | 2 | 25 | 0.1 | 0.1 | 22.8 | 50 | MM2 |
Sample 4 | No Cells | T2 | H3 | p6RC/p12 | 2 | 25 | 0.1 | 0.1 | 22.8 | 50 | MM2 |
NTC | No qPCR sample | NA | A12 | p24RC/p4 | 0 | 25 | 0.1 | 0.1 | 24.8 | 50 | MM1 |
NTC | No qPCR sample | NA | B12 | p6RC/p12 | 0 | 25 | 0.1 | 0.1 | 24.8 | 50 | MM2 |
Results[edit]
- 20161024-qPCR-CellTest3-T1-Repeat.png
qPCR Results T1; After C Probes Before Wash
- 20161024-qPCR-CellTest3-T2-Repeat.png
qPCR Results T2; After C Probes After Wash
- 2016-10-24-CellTest3-TimePoints-T1-Repeat.png
Gel Image 1 Repeat; After C Probes Before Wash
- 2016-10-24-CellTest3-TimePoints-T2-Repeat.png
Gel Image 2 Repeat; After C Probes After Wash
Additional Analysis[edit]
I can test the circular product existence by replacing AmpF with primer12, as before.
- qPCR
- Prepare the following master mix
- 107.1 uL nfH2O
- 5.1 uL 10 uM primer 12
- 127.5 uL Kapa SYBR Fast
- Add 43 uL master mix to each well
- Add 2 uL sample and 5 uL correct AmpR Index (10 uM) to correct well by following sample 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
- 20161024-qPCR-CellTest3-primer12AmpR.png
qPCR Curve
- 2016-10-25-CellTest3-p12rc-AmpR.png
Gel Image
Sample | Condition | Lane | AmpR Index | Sample Vol (uL) | 2X Kapa SYBR qPCR MM | 10 uM primer 12 | 10 uM AmpR6.3-IndXX | H2O | Total Volume (uL) |
Sample 1 | Exp. Rep 1 | A1 | 20 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2 | Exp. Rep 2 | B1 | 20 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3 | No C Probes | C1 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4 | No Cells | D1 | 22 | 2 | 25 | 1 | 1 | 21 | 50 |
NTC | No qPCR sample | E1 | 23 | 0 | 25 | 1 | 1 | 23 | 50 |