Daniel:Notebook/ComboLock/2017-1-26: Difference between revisions
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>Djacobse |
>Djacobse |
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<li>Add 47 uL master mix to each well</li> | <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> | <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="100" height="45" | Sample | |||
| width="100" | Condition | |||
| width="65" | Lane | |||
| width="65" | AmpR Index | |||
| width="65" | Sample Vol (uL) | |||
| width="85" | 2X Kapa SYBR qPCR MM | |||
| width="65" | 10 uM Forward Primer | |||
| width="65" | 10 uM Reverse Primer | |||
| width="65" | H2O | |||
| width="65" | Total Volume (uL) | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 6AC | |||
| align="center" | Epicentre | |||
| align="center" | A1 | |||
| 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 6BC | |||
| align="center" | Epicentre | |||
| align="center" | A2 | |||
| 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 6AD | |||
| align="center" | Epicentre 1:10 | |||
| align="center" | A3 | |||
| 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" valign="bottom" | Sample 6BD | |||
| align="center" | Epicentre 1:10 | |||
| align="center" | A4 | |||
| 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" valign="bottom" | NTC | |||
| align="center" valign="bottom" | | |||
| align="center" valign="bottom" | A5 | |||
| align="center" align="center" valign="bottom" | 24 | |||
| align="center" align="center" valign="bottom" | 2 | |||
| align="center" align="center" valign="bottom" | 25 | |||
| align="center" align="center" valign="bottom" | 1 | |||
| align="center" align="center" valign="bottom" | 1 | |||
| align="center" align="center" valign="bottom" | 21 | |||
| align="center" align="center" valign="bottom" | 50 | |||
|} | |||
<li>qPCR Cycles</li> | <li>qPCR Cycles</li> |
Revision as of 21:10, 26 January 2017
RCA Test (Started Monday)
Epicentre Test (Started Yesterday)
Sample Matrix
- qPCR
- Make 5.2X qPCR master mix according to following recipe
- 109.2 uL nfH2o
- 5.2 uL AmpF</li
- 130 uL 2X Kapa SYBR Fast Master Mix
- 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 64 uL TBE, 16 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
- 20170126-qPCR-RCATest-Epicentre.png
qPCR curve
- 2017-01-26-RCATest-Epicentre.png
Gel image
Sample | Condition | Lane | AmpR Index | Sample Vol (uL) | 2X Kapa SYBR qPCR MM | 10 uM Forward Primer | 10 uM Reverse Primer | H2O | Total Volume (uL) |
Sample 6AC | Epicentre | A1 | 22 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 6BC | Epicentre | A2 | 22 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 6AD | Epicentre 1:10 | A3 | 23 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 6BD | Epicentre 1:10 | A4 | 23 | 2 | 25 | 1 | 1 | 21 | 50 |
NTC | A5 | 24 | 2 | 25 | 1 | 1 | 21 | 50 |