Daniel:Notebook/ComboLock/2016-5-25
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C Probe Hybridization test (VIM, Started 5/23/2016)[edit]
From yesterday's results, I'm going to try the qPCR again. I saved the samples in the 4C, so I just went straight to the qPCR. Hopefully this time I will get more conclusive evidence one way or the other.
Protocol[edit]
- 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 |
Results[edit]
Row | Sample | Cq |
A01 | Sample 1 (C1) | 15.16 |
B01 | Sample 2 (C2) | 13.55 |
C01 | Sample 3 (C1+C2) | 13.45 |
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 |
File:CProbeTest Results2 20160525 VIM.png
Discussion[edit]
Once again all the negative controls failed and so did the positive control. However, this time all 3 samples amplified correctly. I'm inclined therefore to call this a successful run, which means I should try the other samples.
Prep for VIM Antibody Test[edit]
Tomorrow I plan on beginning the anitbody prep for testing VIM. Today there is some prep work to be done.
- Prep 100 mL of sodium phosphate buffer
- Make 100 mL of NaH2PO4 (monobasic) by adding 13.8g to nfH2O
- Make 100 mL of Na2H2PO4 (dibasic) by adding 14.2g to nfH2O
- Mix 28 mL of monobasic and 72 mL of dibasic to make 1 M (buffer pH 7.2)
- Directions from Cold Spring Harbor
- Sulfo-SMCC
- Dissolve 50 mg sulfo-SMCC in 990 uL nf H20 and 110 uL 1M phosphate buffer (Conc: 100 mM SulfoSMCC)
- Aliquot in 100 uL increments
- Store at -20C with dessicant