Daniel:Notebook/ComboLock/2017-2-8: Difference between revisions
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= | =v3 Oligos-Round 3= | ||
[[Daniel:Notebook/ComboLock|Back to Calendar]] | |||
==Protocol== | |||
<ol start="9"> | |||
<li>qPCR</li> | |||
<ol type="A"> | |||
<li>Make 21.2X qPCR master mix according to following recipe</li> | |||
<ol type="a"> | |||
<li>445.2 uL nfH2O</li> | |||
<li>530 uL SYBR Fast</li> | |||
<li>21.2 uL 10 uM AmpF</li> | |||
</ol> | |||
<li>Add 47 uL master mix to each well</li> | |||
<li>Add 2 uL sample and 1 uL appropriate Reverse Primer (1 uM) according to table</li> | |||
[[ | {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | ||
|- style="background-color:#92CDDC;font-size:12pt;font-weight:bold" align="center" | |||
| width="110" height="45" | Sample | |||
| width="180" | Condition | |||
| width="65" | Pre/Post RCA | |||
| width="65" | Lane | |||
| width="65" | AmpR Index | |||
| width="65" | Sample Vol (uL) | |||
| width="95" | 2X Kapa SYBR qPCR MM | |||
| width="95" | 10 uM Forward Primer | |||
| width="95" | 10 uM Reverse Primer | |||
| width="65" | H2O | |||
| width="65" | Total Volume (uL) | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 1A | |||
| align="center" | Sample | |||
| align="center" | Pre | |||
| align="center" | A1 | |||
| align="center" align="center" | 20 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 1B | |||
| align="center" | Sample | |||
| align="center" | Pre | |||
| align="center" | A2 | |||
| align="center" align="center" | 20 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 2A | |||
| align="center" | No Template | |||
| align="center" | Pre | |||
| align="center" | A3 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | Sample 2B | |||
| align="center" valign="bottom" | No Template | |||
| align="center" valign="bottom" | Pre | |||
| align="center" | A4 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 3A | |||
| align="center" | No C Probe | |||
| align="center" | Pre | |||
| align="center" | A5 | |||
| align="center" align="center" | 22 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 3B | |||
| align="center" | No C Probe | |||
| align="center" | Pre | |||
| align="center" | A6 | |||
| align="center" align="center" | 22 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 4A | |||
| align="center" | No Lock Phosphate | |||
| align="center" | Pre | |||
| align="center" | A7 | |||
| align="center" align="center" | 23 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 4B | |||
| align="center" | No Lock Phosphate | |||
| align="center" valign="bottom" | Pre | |||
| align="center" | A8 | |||
| align="center" align="center" | 23 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 1AX | |||
| align="center" | Sample | |||
| align="center" | Post | |||
| align="center" | H1 | |||
| align="center" align="center" | 24 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="background-color:#DA9694;font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 1BX | |||
| align="center" | Sample | |||
| align="center" | Post | |||
| align="center" | H2 | |||
| align="center" align="center" | 24 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 2AX | |||
| align="center" | No Template | |||
| align="center" | Post | |||
| align="center" | H3 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="background-color:#DA9694;font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | Sample 2BX | |||
| align="center" valign="bottom" | No Template | |||
| align="center" valign="bottom" | Post | |||
| align="center" | H4 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 3AX | |||
| align="center" | No C Probe | |||
| align="center" | Post | |||
| align="center" | H5 | |||
| align="center" align="center" | 26 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="background-color:#DA9694;font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 3BX | |||
| align="center" | No C Probe | |||
| align="center" | Post | |||
| align="center" | H6 | |||
| align="center" align="center" | 26 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 4AX | |||
| align="center" | No Lock Phosphate | |||
| align="center" | Post | |||
| align="center" | H7 | |||
| align="center" align="center" | 27 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="background-color:#DA9694;font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 4BX | |||
| align="center" | No Lock Phosphate | |||
| align="center" valign="bottom" | Post | |||
| align="center" | H8 | |||
| align="center" align="center" | 27 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | NTC | |||
| align="center" valign="bottom" | qPCR Primers Only | |||
| align="center" align="center" valign="bottom" | | |||
| align="center" | D5 | |||
| align="center" align="center" | 30 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 23 | |||
| align="center" align="center" | 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 x24</li> | |||
<li>72C 2 min</li> | |||
<li>16C hold</li> | |||
</ol></ol> | |||
<li>TBE Gel</li> | |||
<ol type="A"> | |||
<li>Mix 224 uL TBE, 56 uL 6x loading dye</li> | |||
<li>Aliquot 10 uL per sample/ladder lane onto parafilm</li> | |||
<li>Add 2 uL of sample or ladder to correct drop</li> | |||
<li>Load 10 uL in to well</li> | |||
<li>Run gel for 23 minutes at 230V</li> | |||
<li>Open gel and stain with 3 uL SYBR Gold for 3 minutes</li> | |||
<li>Rinse gel and image in gel doc</li> | |||
</ol></ol> | |||
===Results=== | |||
<gallery perrow=2 heights=300px widths=300px> | <gallery perrow=2 heights=300px widths=300px> |
Revision as of 20:29, 8 February 2017
v3 Oligos-Round 3
Protocol
- qPCR
- Make 21.2X qPCR master mix according to following recipe
- 445.2 uL nfH2O
- 530 uL SYBR Fast
- 21.2 uL 10 uM AmpF
- Add 47 uL master mix to each well
- Add 2 uL sample and 1 uL appropriate Reverse Primer (1 uM) according to table
- 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 224 uL TBE, 56 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 23 minutes at 230V
- Open gel and stain with 3 uL SYBR Gold for 3 minutes
- Rinse gel and image in gel doc
Sample | Condition | Pre/Post RCA | Lane | AmpR Index | Sample Vol (uL) | 2X Kapa SYBR qPCR MM | 10 uM Forward Primer | 10 uM Reverse Primer | H2O | Total Volume (uL) |
Sample 1A | Sample | Pre | A1 | 20 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1B | Sample | Pre | A2 | 20 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2A | No Template | Pre | A3 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2B | No Template | Pre | A4 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3A | No C Probe | Pre | A5 | 22 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3B | No C Probe | Pre | A6 | 22 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4A | No Lock Phosphate | Pre | A7 | 23 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4B | No Lock Phosphate | Pre | A8 | 23 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1AX | Sample | Post | H1 | 24 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1BX | Sample | Post | H2 | 24 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2AX | No Template | Post | H3 | 25 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2BX | No Template | Post | H4 | 25 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3AX | No C Probe | Post | H5 | 26 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3BX | No C Probe | Post | H6 | 26 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4AX | No Lock Phosphate | Post | H7 | 27 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4BX | No Lock Phosphate | Post | H8 | 27 | 2 | 25 | 1 | 1 | 21 | 50 |
NTC | qPCR Primers Only | D5 | 30 | 2 | 25 | 1 | 1 | 23 | 50 |
Results
- 2017-02-08-v3Oligos-LoTemp-PreRCA.png
Gel image-Pre RCA
- 2017-02-08-v3Oligos-LoTemp-PostRCA.png
Gel image-Post RCA
- 2017-02-08-v3Oligos-LoTemp-PostRCA-Highlight.png
Gel image-Post RCA-Product Highlighted