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)

Back to Calendar

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.

  1. Set up reaction according to table
  2. 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
  3. qPCR Cycles
    1. 95C 3 min
    2. 95C 3 sec
    3. 55C 30 sec
    4. 72C 20 sec
    5. plate read
    6. goto b x20
    7. 72C 2 min
    8. 16C hold


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.