Daniel:Notebook/ComboLock/2016-5-25: Difference between revisions
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[[Daniel:Notebook/ComboLock|Back to Calendar]] | [[Daniel:Notebook/ComboLock|Back to Calendar]] | ||
From [[Daniel:Notebook/ComboLock/2016-5-24|yesterday]]'s results, I'm going to try the qPCR again. Hopefully this time I will get more conclusive evidence one way or the other. | |||
<ol type="A"> | |||
<li>Set up reaction according to table</li> | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#FCD5B4;font-size:12pt" align="center" | |||
| width="175" height="45" | Sample | |||
| width="65" | Lane | |||
| width="65" | Cell Sample | |||
| width="65" | Cells | |||
| width="88" | 2X Kapa SYBR qPCR MM | |||
| width="65" | 100 uM Primer2 | |||
| width="65" | 100 uM Primer4-RC | |||
| width="65" | 100 uM Primer6 | |||
| width="81" | 100 uM Primer12-RC | |||
| width="65" | H2O | |||
| width="65" | Total Volume (uL) | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Sample 1 (C1) | |||
| align="center" valign="bottom" | A1 | |||
| align="center" valign="bottom" | S1 | |||
| align="center" align="center" valign="bottom" | 1 | |||
| align="center" align="center" valign="bottom" | 25 | |||
| align="center" align="center" valign="bottom" | 0.5 | |||
| align="center" align="center" valign="bottom" | 0.5 | |||
| align="center" align="center" valign="bottom" | 0 | |||
| align="center" align="center" valign="bottom" | 0 | |||
| align="center" align="center" valign="bottom" | 23 | |||
| align="center" align="center" valign="bottom" | 50 | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| height="15" valign="bottom" | Sample 2 (C2) | |||
| align="center" valign="bottom" | A2 | |||
| align="center" valign="bottom" | S2 | |||
| align="center" align="center" valign="bottom" | 1 | |||
| align="center" align="center" valign="bottom" | 25 | |||
| align="center" align="center" valign="bottom" | 0 | |||
| align="center" align="center" valign="bottom" | 0 | |||
| align="center" align="center" valign="bottom" | 0.5 | |||
| align="center" align="center" valign="bottom" | 0.5 | |||
| align="center" align="center" valign="bottom" | 23 | |||
| align="center" align="center" valign="bottom" | 50 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Sample 3 (C1+C2) | |||
| align="center" valign="bottom" | A3 | |||
| align="center" valign="bottom" | S3 | |||
| align="center" align="center" valign="bottom" | 1 | |||
| align="center" align="center" valign="bottom" | 25 | |||
| align="center" align="center" valign="bottom" | 0.5 | |||
| align="center" align="center" valign="bottom" | 0.5 | |||
| align="center" align="center" valign="bottom" | 0.5 | |||
| align="center" align="center" valign="bottom" | 0.5 | |||
| align="center" align="center" valign="bottom" | 22 | |||
| align="center" align="center" valign="bottom" | 50 | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| height="15" valign="bottom" | Sample 4 (No Probe) | |||
| align="center" valign="bottom" | A4 | |||
| align="center" valign="bottom" | S4 | |||
| align="center" align="center" valign="bottom" | 1 | |||
| align="center" align="center" valign="bottom" | 25 | |||
| align="center" align="center" valign="bottom" | 0.5 | |||
| align="center" align="center" valign="bottom" | 0.5 | |||
| align="center" align="center" valign="bottom" | 0.5 | |||
| align="center" align="center" valign="bottom" | 0.5 | |||
| align="center" align="center" valign="bottom" | 22 | |||
| align="center" align="center" valign="bottom" | 50 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | No Template | |||
| align="center" valign="bottom" | A5 | |||
| align="center" valign="bottom" | NA | |||
| align="center" align="center" valign="bottom" | 0 | |||
| align="center" align="center" valign="bottom" | 25 | |||
| align="center" align="center" valign="bottom" | 0.5 | |||
| align="center" align="center" valign="bottom" | 0.5 | |||
| align="center" align="center" valign="bottom" | 0.5 | |||
| align="center" align="center" valign="bottom" | 0.5 | |||
| align="center" align="center" valign="bottom" | 23 | |||
| align="center" align="center" valign="bottom" | 50 | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| height="15" valign="bottom" | Mismatch Primers | |||
| align="center" valign="bottom" | A6 | |||
| align="center" valign="bottom" | S3 | |||
| align="center" align="center" valign="bottom" | 1 | |||
| align="center" align="center" valign="bottom" | 25 | |||
| align="center" align="center" valign="bottom" | 0.5 | |||
| align="center" align="center" valign="bottom" | 0 | |||
| align="center" align="center" valign="bottom" | 0.5 | |||
| align="center" align="center" valign="bottom" | 0 | |||
| align="center" align="center" valign="bottom" | 23 | |||
| align="center" align="center" valign="bottom" | 50 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | No Primers | |||
| align="center" valign="bottom" | A7 | |||
| align="center" valign="bottom" | S3 | |||
| align="center" align="center" valign="bottom" | 1 | |||
| align="center" align="center" valign="bottom" | 25 | |||
| align="center" align="center" valign="bottom" | 0 | |||
| align="center" align="center" valign="bottom" | 0 | |||
| align="center" align="center" valign="bottom" | 0 | |||
| align="center" align="center" valign="bottom" | 0 | |||
| align="center" align="center" valign="bottom" | 24 | |||
| align="center" align="center" valign="bottom" | 50 | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| height="15" valign="bottom" | Primers + C Probes | |||
| align="center" valign="bottom" | A8 | |||
| align="center" valign="bottom" | NA | |||
| align="center" align="center" valign="bottom" | 1 | |||
| align="center" align="center" valign="bottom" | 25 | |||
| align="center" align="center" valign="bottom" | 0.5 | |||
| align="center" align="center" valign="bottom" | 0.5 | |||
| align="center" align="center" valign="bottom" | 0.5 | |||
| align="center" align="center" valign="bottom" | 0.5 | |||
| align="center" align="center" valign="bottom" | 22 | |||
| align="center" align="center" valign="bottom" | 50 | |||
|} | |||
<li>qPCR Cycles</li> | |||
<ol type="a"> | |||
<li>95C 3 min</li> | |||
<li>95C 3 sec</li> | |||
<li>55C 30 sec</li> | |||
<li>72C 20 sec</li> | |||
<li>plate read</li> | |||
<li>goto b x20</li> | |||
<li>72C 2 min</li> | |||
<li>16C hold</li> | |||
</ol> | |||
</ol> | |||
</ol> | |||
===Results=== | ===Results=== |
Revision as of 18:22, 25 May 2016
C Probe Hybridization test (VIM, Started 5/23/2016)
From yesterday's results, I'm going to try the qPCR again. Hopefully this time I will get more conclusive evidence one way or the other.
- 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
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
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.