Daniel:Notebook/ComboLock/2016-9-26: Difference between revisions
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>Djacobse |
>Djacobse |
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==Protocol== | ==Protocol== | ||
<ol start=" | <ol start="5"> | ||
<li>qPCR</li> | <li>qPCR</li> | ||
<ol type="A"> | <ol type="A"> | ||
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|- style="background-color:#D9D9D9;font-size:12pt" | |- style="background-color:#D9D9D9;font-size:12pt" | ||
|style="font-weight:bold" height="15" valign="bottom" | Sample 1A | |style="font-weight:bold" height="15" valign="bottom" | Sample 1A | ||
| align="center" align="center" valign="bottom" | 1.00E+ | | align="center" align="center" valign="bottom" | 1.00E+09 | ||
| align="center" valign="bottom" | A1 | | align="center" valign="bottom" | A1 | ||
| align="center" align="center" valign="bottom" | 36 | | align="center" align="center" valign="bottom" | 36 | ||
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|- style="font-size:12pt" | |- style="font-size:12pt" | ||
|style="font-weight:bold" height="15" valign="bottom" | Sample 1B | |style="font-weight:bold" height="15" valign="bottom" | Sample 1B | ||
| align="center" align="center" valign="bottom" | 1.00E+ | | align="center" align="center" valign="bottom" | 1.00E+09 | ||
| align="center" valign="bottom" | A2 | | align="center" valign="bottom" | A2 | ||
| align="center" align="center" valign="bottom" | 36 | | align="center" align="center" valign="bottom" | 36 | ||
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|- style="background-color:#D9D9D9;font-size:12pt" | |- style="background-color:#D9D9D9;font-size:12pt" | ||
|style="font-weight:bold" height="15" valign="bottom" | Sample 1C | |style="font-weight:bold" height="15" valign="bottom" | Sample 1C | ||
| align="center" align="center" valign="bottom" | 1.00E+ | | align="center" align="center" valign="bottom" | 1.00E+09 | ||
| align="center" valign="bottom" | A3 | | align="center" valign="bottom" | A3 | ||
| align="center" align="center" valign="bottom" | 36 | | align="center" align="center" valign="bottom" | 36 | ||
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|- style="background-color:#D8E4BC;font-size:12pt" | |- style="background-color:#D8E4BC;font-size:12pt" | ||
|style="font-weight:bold" height="15" valign="bottom" | Sample 1A | |style="font-weight:bold" height="15" valign="bottom" | Sample 1A | ||
| align="center" align="center" valign="bottom" | 1.00E+ | | align="center" align="center" valign="bottom" | 1.00E+09 | ||
| align="center" valign="bottom" | H1 | | align="center" valign="bottom" | H1 | ||
| align="center" align="center" valign="bottom" | 36 | | align="center" align="center" valign="bottom" | 36 | ||
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|- style="font-size:12pt" | |- style="font-size:12pt" | ||
|style="font-weight:bold" height="15" valign="bottom" | Sample 1B | |style="font-weight:bold" height="15" valign="bottom" | Sample 1B | ||
| align="center" align="center" valign="bottom" | 1.00E+ | | align="center" align="center" valign="bottom" | 1.00E+09 | ||
| align="center" valign="bottom" | H2 | | align="center" valign="bottom" | H2 | ||
| align="center" align="center" valign="bottom" | 36 | | align="center" align="center" valign="bottom" | 36 | ||
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|- style="background-color:#D8E4BC;font-size:12pt" | |- style="background-color:#D8E4BC;font-size:12pt" | ||
|style="font-weight:bold" height="15" valign="bottom" | Sample 1C | |style="font-weight:bold" height="15" valign="bottom" | Sample 1C | ||
| align="center" align="center" valign="bottom" | 1.00E+ | | align="center" align="center" valign="bottom" | 1.00E+09 | ||
| align="center" valign="bottom" | H3 | | align="center" valign="bottom" | H3 | ||
| align="center" align="center" valign="bottom" | 36 | | align="center" align="center" valign="bottom" | 36 | ||
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|- style="background-color:#FCD5B4;font-size:12pt" | |- style="background-color:#FCD5B4;font-size:12pt" | ||
|style="font-weight:bold" height="15" valign="bottom" | Sample 1A | |style="font-weight:bold" height="15" valign="bottom" | Sample 1A | ||
| align="center" align="center" valign="bottom" | 1.00E+ | | align="center" align="center" valign="bottom" | 1.00E+09 | ||
| align="center" valign="bottom" | C1 | | align="center" valign="bottom" | C1 | ||
| align="center" align="center" valign="bottom" | 37 | | align="center" align="center" valign="bottom" | 37 | ||
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|- style="font-size:12pt" | |- style="font-size:12pt" | ||
|style="font-weight:bold" height="15" valign="bottom" | Sample 1B | |style="font-weight:bold" height="15" valign="bottom" | Sample 1B | ||
| align="center" align="center" valign="bottom" | 1.00E+ | | align="center" align="center" valign="bottom" | 1.00E+09 | ||
| align="center" valign="bottom" | C2 | | align="center" valign="bottom" | C2 | ||
| align="center" align="center" valign="bottom" | 37 | | align="center" align="center" valign="bottom" | 37 | ||
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|- style="background-color:#FCD5B4;font-size:12pt" | |- style="background-color:#FCD5B4;font-size:12pt" | ||
|style="font-weight:bold" height="15" valign="bottom" | Sample 1C | |style="font-weight:bold" height="15" valign="bottom" | Sample 1C | ||
| align="center" align="center" valign="bottom" | 1.00E+ | | align="center" align="center" valign="bottom" | 1.00E+09 | ||
| align="center" valign="bottom" | C3 | | align="center" valign="bottom" | C3 | ||
| align="center" align="center" valign="bottom" | 37 | | align="center" align="center" valign="bottom" | 37 | ||
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|} | |} | ||
<li>Make master mixes according to following table</li> | |||
<li>Add 48 uL correct master mix to each well</li> | <li>Add 48 uL correct master mix to each well</li> |
Revision as of 18:34, 26 September 2016
PCR Testing (Started Thursday)
Continuing the tests on qPCR for the positive control amplicon using primer concentrations. This is following some of the conclusions from Friday.
Protocol
- qPCR
- Follow reaction map below
- Make master mixes according to following table
- Add 48 uL correct master mix to each well
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x26
- 72C 2 min
- 16C hold
Sample | Molecule Count | Lane | AmpR Index | Sample Vol (uL) | 2X Kapa SYBR qPCR MM | 10 uM AmpF 6.4 | 10 uM AmpR6.3-IndXX | H2O | Total Volume (uL) | Master Mix |
Sample 1A | 1.00E+09 | A1 | 36 | 2 | 25 | 5 | 5 | 13 | 50 | MM1 |
Sample 1B | 1.00E+09 | A2 | 36 | 2 | 25 | 5 | 5 | 13 | 50 | MM1 |
Sample 1C | 1.00E+09 | A3 | 36 | 2 | 25 | 5 | 5 | 13 | 50 | MM1 |
NTC | 0 | A4 | 36 | 0 | 25 | 5 | 5 | 13 | 50 | NTC |
Sample 1A | 1.00E+09 | A5 | 36 | 2 | 25 | 1 | 1 | 21 | 50 | MM2 |
Sample 1B | 1.00E+09 | A6 | 36 | 2 | 25 | 1 | 1 | 21 | 50 | MM2 |
Sample 1C | 1.00E+09 | A7 | 36 | 2 | 25 | 1 | 1 | 21 | 50 | MM2 |
NTC | 0 | A8 | 36 | 0 | 25 | 1 | 1 | 19.5 | 50 | NTC |
Sample 1A | 1.00E+09 | H1 | 36 | 2 | 25 | 0.5 | 0.5 | 22 | 50 | MM3 |
Sample 1B | 1.00E+09 | H2 | 36 | 2 | 25 | 0.5 | 0.5 | 22 | 50 | MM3 |
Sample 1C | 1.00E+09 | H3 | 36 | 2 | 25 | 0.5 | 0.5 | 22 | 50 | MM3 |
NTC | 0 | H4 | 36 | 0 | 25 | 0.5 | 0.5 | 19.5 | 50 | NTC |
Sample 1A | 1.00E+09 | H5 | 37 | 2 | 25 | 0.1 | 0.1 | 22.8 | 50 | MM4 |
Sample 1B | 1.00E+09 | H6 | 37 | 2 | 25 | 0.1 | 0.1 | 22.8 | 50 | MM4 |
Sample 1C | 1.00E+09 | H7 | 37 | 2 | 25 | 0.1 | 0.1 | 22.8 | 50 | MM4 |
NTC | 0 | H8 | 37 | 0 | 25 | 0.1 | 0.1 | 19.5 | 50 | NTC |
Sample 1A | 1.00E+09 | C1 | 37 | 2 | 25 | 0.01 | 0.01 | 23 | 50 | MM5 |
Sample 1B | 1.00E+09 | C2 | 37 | 2 | 25 | 0.01 | 0.01 | 23 | 50 | MM5 |
Sample 1C | 1.00E+09 | C3 | 37 | 2 | 25 | 0.01 | 0.01 | 23 | 50 | MM5 |
NTC | 0 | C4 | 37 | 0 | 25 | 0.01 | 0.01 | 19.5 | 50 | NTC |