Daniel:Notebook/ComboLock/2016-5-24
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C Probe Hybridization Test (VIM, Prep yesterday)[edit]
Protocol[edit]
Sample Matrix[edit]
Sample | VIM-C1 (1 uL) | VIM-C2 (1 uL) |
Sample 1 | X | |
Sample 2 | X | |
Sample 3 | X | X |
Sample 4 |
- Hybridization (Lab)
- Split cells into 4 samples (1.25 mL methanol each)
- Pellet cells via centrifugation at 600g for 3 min
- Put 1 uL C1 Probe and 1 uL C2 probe into separate 0.2 mL tubes
- Heat C probes to 90C for 5 min; Chill probes on ice to quench
- Add probes to hybridization buffer for final concentration of 100 nM (0.5 uL/probe/sample using 0.5mL hybridization buffer)
- Incubate probes at 40C for 1 hour with vigorous agitation
- Wash three times with 0.5 mL wash buffer and pelleting at 600xg for 3 min
- qPCR
- Set up reaction according to table
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x20
- 72C 2 min
- 16C hold
Sample | Lane | Cell Sample | Cells | 2X Kapa SYBR qPCR MM | 100 uM Primer2 | 100 uM Primer4-RC | 100 uM Primer6 | 100 uM Primer12-RC | H2O | Total Volume (uL) |
Sample 1 (C1) | A1 | S1 | 1 | 25 | 0.5 | 0.5 | 0 | 0 | 23 | 50 |
Sample 2 (C2) | A2 | S2 | 1 | 25 | 0 | 0 | 0.5 | 0.5 | 23 | 50 |
Sample 3 (C1+C2) | A3 | S3 | 1 | 25 | 0.5 | 0.5 | 0.5 | 0.5 | 22 | 50 |
Sample 4 (No Probe) | A4 | S4 | 1 | 25 | 0.5 | 0.5 | 0.5 | 0.5 | 22 | 50 |
No Template | A5 | NA | 0 | 25 | 0.5 | 0.5 | 0.5 | 0.5 | 23 | 50 |
Mismatch Primers | A6 | S3 | 1 | 25 | 0.5 | 0 | 0.5 | 0 | 23 | 50 |
No Primers | A7 | S3 | 1 | 25 | 0 | 0 | 0 | 0 | 24 | 50 |
Primers + C Probes | A8 | NA | 1 | 25 | 0.5 | 0.5 | 0.5 | 0.5 | 22 | 50 |
Buffers[edit]
- Wash Buffer: 1X PBS, 0.1% Tween-20, 4 U/mL RNasin
- Hybridization Buffer: 1X SSC, 20mM RVC, 40 U/mL RNasin, 0.1% Tween 20, 100 ug/mL salmon sperm DNA
qPCR Results[edit]
Row | Sample | Cq |
A01 | Sample 1 (C1) | N/A |
B01 | Sample 2 (C2) | 13.1 |
C01 | Sample 3 (C1+C2) | 12.99 |
D01 | Sample 4 (No Probe) | N/A |
E01 | No Template | N/A |
F01 | Mismatch Primers | N/A |
G01 | No Primers | N/A |
H01 | Primers + C Probes | N/A |
Discussion[edit]
The negative controls were all negative, which is good. However, the positive control showed no results and only 2 of 3 samples amplified. I think it would be best to repeat the experiment tomorrow.