Daniel:Notebook/ComboLock/2016-10-25
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Cell Test 3 (Started Friday 10/21)[edit]
Latch-Padlock Detection qPCR[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 200 uL TBE, 50 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 | Target | Forward Primer 10 uM (uL) | Reverse Primer10 uM (uL) | nfH2O (uL) | Kapa SYBR Fast MM (uL) |
MM1 | p24RC/p12 | Padlock0201 | 10.1 | 10.1 | 212.1 | 252.5 |
MM2 | p4/p6RC | Latch0001 | 10.1 | 10.1 | 212.1 | 252.5 |
qPCR Sample Table[edit]
Sample | Condition | Time Point | Lane | Latch/Padlock Target | Sample Vol (uL) | 2X Kapa SYBR qPCR MM | 10 uM Forward Primer | 10 uM Reverse Primer | H2O | Total Volume (uL) | Master Mix |
Sample 1 | Exp. Rep 1 | T3 | A1 | Padlock | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 2 | Exp. Rep 2 | T3 | B1 | Padlock | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 3 | No C Probes | T3 | C1 | Padlock | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 4 | No Cells | T3 | D1 | Padlock | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 1 | Exp. Rep 1 | T3 | E1 | Latch | 2 | 25 | 1 | 1 | 21 | 50 | MM2 |
Sample 2 | Exp. Rep 2 | T3 | F1 | Latch | 2 | 25 | 1 | 1 | 21 | 50 | MM2 |
Sample 3 | No C Probes | T3 | G1 | Latch | 2 | 25 | 1 | 1 | 21 | 50 | MM2 |
Sample 4 | No Cells | T3 | H1 | Latch | 2 | 25 | 1 | 1 | 21 | 50 | MM2 |
Sample 1 | Exp. Rep 1 | T4 | A2 | Padlock | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 2 | Exp. Rep 2 | T4 | B2 | Padlock | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 3 | No C Probes | T4 | C2 | Padlock | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 4 | No Cells | T4 | D2 | Padlock | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
Sample 1 | Exp. Rep 1 | T4 | E2 | Latch | 2 | 25 | 1 | 1 | 21 | 50 | MM2 |
Sample 2 | Exp. Rep 2 | T4 | F2 | Latch | 2 | 25 | 1 | 1 | 21 | 50 | MM2 |
Sample 3 | No C Probes | T4 | G2 | Latch | 2 | 25 | 1 | 1 | 21 | 50 | MM2 |
Sample 4 | No Cells | T4 | H2 | Latch | 2 | 25 | 1 | 1 | 21 | 50 | MM2 |
NTC | No qPCR sample | NA | A12 | Padlock | 0 | 25 | 1 | 1 | 23 | 50 | MM1 |
NTC | No qPCR sample | NA | B12 | Latch | 0 | 25 | 1 | 1 | 23 | 50 | MM2 |
PC-Padlock | No qPCR sample | NA | C12 | Padlock | 2 | 25 | 1 | 1 | 21 | 50 | MM1 |
PC-Latch | No qPCR sample | NA | D12 | Latch | 2 | 25 | 1 | 1 | 21 | 50 | MM2 |
Results[edit]
- 20161025-qPCR-CellTest3-T3-LatchPadlock.png
qPCR Curve T3, after LP hybridization before wash
- 20161025-qPCR-CellTest3-T4-LatchPadlock.png
qPCR Curve T4, after LP hybridization after wash
- 2016-10-25-CellTest3-T3-LatchPadlock.png
Gel Image T3, after LP hybridization before wash
- 2016-10-25-CellTest3-T4-LatchPadlock.png
Gel Image T4, after LP hybridization after wash