Daniel:Notebook/ComboLock/2016-9-26: Difference between revisions

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>Djacobse
>Djacobse
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==Protocol==
==Protocol==


<ol start="4">
<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+10
| 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+10
| 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+10
| 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+10
| 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+10
| 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+10
| 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+10
| 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
Line 249: Line 249:
|- 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+10
| 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+10
| 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)

Back to Calendar

Continuing the tests on qPCR for the positive control amplicon using primer concentrations. This is following some of the conclusions from Friday.

Protocol

  1. qPCR
    1. Follow reaction map below
    2. 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
    3. Make master mixes according to following table
    4. Add 48 uL correct master mix to each well
    5. qPCR Cycles
      1. 95C 3 min
      2. 95C 3 sec
      3. 55C 30 sec
      4. 72C 20 sec
      5. plate read
      6. goto b x26
      7. 72C 2 min
      8. 16C hold