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

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[[Daniel:Notebook/ComboLock|Back to Calendar]]
[[Daniel:Notebook/ComboLock|Back to Calendar]]


Continuing the tests on qPCR for the positive control amplicon using primer concentrations. This is following some of the conclusions from [[Daniel:Notebook/ComboLock/216-9-23|Friday]].
Continuing the tests on qPCR for the positive control amplicon using primer concentrations. This is following some of the conclusions from [[Daniel:Notebook/ComboLock/2016-9-23|Friday]].


==Protocol==
==Protocol==
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| align="center" align="center" valign="bottom" | 1
| align="center" align="center" valign="bottom" | 1
| align="center" align="center" valign="bottom" | 1
| align="center" align="center" valign="bottom" | 1
| align="center" align="center" valign="bottom" | 19.5
| align="center" align="center" valign="bottom" | 21
| align="center" align="center" valign="bottom" | 50
| align="center" align="center" valign="bottom" | 50
| align="center" valign="bottom" | NTC
| align="center" valign="bottom" | NTC
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| 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" | 19.5
| align="center" align="center" valign="bottom" | 22
| align="center" align="center" valign="bottom" | 50
| align="center" align="center" valign="bottom" | 50
| align="center" valign="bottom" | NTC
| align="center" valign="bottom" | NTC
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| align="center" align="center" valign="bottom" | 2
| align="center" align="center" valign="bottom" | 2
| align="center" align="center" valign="bottom" | 25
| align="center" align="center" valign="bottom" | 25
| align="center" align="center" valign="bottom" | 0.1
| align="center" valign="bottom" | 2*
| align="center" align="center" valign="bottom" | 0.1
| align="center" valign="bottom" | 2*
| align="center" align="center" valign="bottom" | 22.8
| align="center" align="center" valign="bottom" | 19
| align="center" align="center" valign="bottom" | 50
| align="center" align="center" valign="bottom" | 50
| align="center" valign="bottom" | MM4
| align="center" valign="bottom" | MM4
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| align="center" align="center" valign="bottom" | 2
| align="center" align="center" valign="bottom" | 2
| align="center" align="center" valign="bottom" | 25
| align="center" align="center" valign="bottom" | 25
| align="center" align="center" valign="bottom" | 0.1
| align="center" valign="bottom" | 2*
| align="center" align="center" valign="bottom" | 0.1
| align="center" valign="bottom" | 2*
| align="center" align="center" valign="bottom" | 22.8
| align="center" align="center" valign="bottom" | 19
| align="center" align="center" valign="bottom" | 50
| align="center" align="center" valign="bottom" | 50
| align="center" valign="bottom" | MM4
| align="center" valign="bottom" | MM4
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| align="center" align="center" valign="bottom" | 2
| align="center" align="center" valign="bottom" | 2
| align="center" align="center" valign="bottom" | 25
| align="center" align="center" valign="bottom" | 25
| align="center" align="center" valign="bottom" | 0.1
| align="center" valign="bottom" | 2*
| align="center" align="center" valign="bottom" | 0.1
| align="center" valign="bottom" | 2*
| align="center" align="center" valign="bottom" | 22.8
| align="center" align="center" valign="bottom" | 19
| align="center" align="center" valign="bottom" | 50
| align="center" align="center" valign="bottom" | 50
| align="center" valign="bottom" | MM4
| align="center" valign="bottom" | MM4
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| align="center" align="center" valign="bottom" | 0
| align="center" align="center" valign="bottom" | 0
| align="center" align="center" valign="bottom" | 25
| align="center" align="center" valign="bottom" | 25
| align="center" align="center" valign="bottom" | 0.1
| align="center" valign="bottom" | 2*
| align="center" align="center" valign="bottom" | 0.1
| align="center" valign="bottom" | 2*
| align="center" align="center" valign="bottom" | 19.5
| align="center" align="center" valign="bottom" | 19
| align="center" align="center" valign="bottom" | 50
| align="center" align="center" valign="bottom" | 50
| align="center" valign="bottom" | NTC
| align="center" valign="bottom" | NTC
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| align="center" align="center" valign="bottom" | 2
| align="center" align="center" valign="bottom" | 2
| align="center" align="center" valign="bottom" | 25
| align="center" align="center" valign="bottom" | 25
| align="center" align="center" valign="bottom" | 0.01
| align="center" valign="bottom" | 0.2*
| align="center" align="center" valign="bottom" | 0.01
| align="center" valign="bottom" | 0.2*
| align="center" align="center" valign="bottom" | 23
| align="center" align="center" valign="bottom" | 22.6
| align="center" align="center" valign="bottom" | 50
| align="center" align="center" valign="bottom" | 50
| align="center" valign="bottom" | MM5
| align="center" valign="bottom" | MM5
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| align="center" align="center" valign="bottom" | 2
| align="center" align="center" valign="bottom" | 2
| align="center" align="center" valign="bottom" | 25
| align="center" align="center" valign="bottom" | 25
| align="center" align="center" valign="bottom" | 0.01
| align="center" valign="bottom" | 0.2*
| align="center" align="center" valign="bottom" | 0.01
| align="center" valign="bottom" | 0.2*
| align="center" align="center" valign="bottom" | 23
| align="center" align="center" valign="bottom" | 22.6
| align="center" align="center" valign="bottom" | 50
| align="center" align="center" valign="bottom" | 50
| align="center" valign="bottom" | MM5
| align="center" valign="bottom" | MM5
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| align="center" align="center" valign="bottom" | 2
| align="center" align="center" valign="bottom" | 2
| align="center" align="center" valign="bottom" | 25
| align="center" align="center" valign="bottom" | 25
| align="center" align="center" valign="bottom" | 0.01
| align="center" valign="bottom" | 0.2*
| align="center" align="center" valign="bottom" | 0.01
| align="center" valign="bottom" | 0.2*
| align="center" align="center" valign="bottom" | 23
| align="center" align="center" valign="bottom" | 22.6
| align="center" align="center" valign="bottom" | 50
| align="center" align="center" valign="bottom" | 50
| align="center" valign="bottom" | MM5
| align="center" valign="bottom" | MM5
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| align="center" align="center" valign="bottom" | 0
| align="center" align="center" valign="bottom" | 0
| align="center" align="center" valign="bottom" | 25
| align="center" align="center" valign="bottom" | 25
| align="center" align="center" valign="bottom" | 0.01
| align="center" valign="bottom" | 0.2*
| align="center" align="center" valign="bottom" | 0.01
| align="center" valign="bottom" | 0.2*
| align="center" align="center" valign="bottom" | 19.5
| align="center" align="center" valign="bottom" | 22.6
| align="center" align="center" valign="bottom" | 50
| align="center" align="center" valign="bottom" | 50
| align="center" valign="bottom" | NTC
| align="center" valign="bottom" | NTC
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<li>Make master mixes according to following table</li>
<li>Make master mixes according to following table</li>
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="background-color:#B8CCE4;font-size:12pt;font-weight:bold" align="center" valign="bottom"
| width="140" rowspan="2" height="15" | Reagent
| width="65" colspan="5" | Master Mix
|- style="background-color:#B8CCE4;font-size:12pt;font-weight:bold" align="center" valign="bottom"
| MM1
| MM2
| MM3
| MM4
| MM5
|- style="font-size:12pt"
| height="15"  valign="bottom" | PrimerF (10 uM)
| align="center" align="center" valign="bottom" | 15.5
| align="center" align="center" valign="bottom" | 3.1
| align="center" align="center" valign="bottom" | 1.55
| align="center" valign="bottom" | 6.2*
| align="center" valign="bottom" | 0.62*
|- style="background-color:#D9D9D9;font-size:12pt"
| height="15"  valign="bottom" | PrimerR (10 uM)
| align="center" align="center" valign="bottom" | 15.5
| align="center" align="center" valign="bottom" | 3.1
| align="center" align="center" valign="bottom" | 1.55
| align="center" valign="bottom" | 6.2*
| align="center" valign="bottom" | 0.62*
|- style="font-size:12pt"
| height="15"  valign="bottom" | KAPA Master Mix
| align="center" align="center" valign="bottom" | 77.5
| align="center" align="center" valign="bottom" | 77.5
| align="center" align="center" valign="bottom" | 77.5
| align="center" align="center" valign="bottom" | 77.5
| align="center" align="center" valign="bottom" | 77.5
|- style="background-color:#D9D9D9;font-size:12pt"
| height="15"  valign="bottom" | nfH2O
| align="center" align="center" valign="bottom" | 40.3
| align="center" align="center" valign="bottom" | 65.1
| align="center" align="center" valign="bottom" | 68.2
| align="center" align="center" valign="bottom" | 58.9
| align="center" align="center" valign="bottom" | 70.06
|}


<li>Add 48 uL correct master mix to each well</li>
<li>Add 48 uL correct master mix to each well</li>
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<li>95C 3 min</li>
<li>95C 3 min</li>
<li>95C 3 sec</li>
<li>95C 3 sec</li>
<li>55C 30 sec</li>
<li>60C 30 sec</li>
<li>72C 20 sec</li>
<li>72C 20 sec</li>
<li>plate read</li>
<li>plate read</li>
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<li>16C hold</li>
<li>16C hold</li>
</ol></ol>
</ol></ol>
===Results===
<gallery perrow=2 heights=300px widths=300px>
File:2016-09-26-PosConPrimersA.png|First Gel
File:2016-09-26-PosConPrimersB.png|Second Gel
File:20160926qPCR_PrimerConc.png|qPCR Curve; averaged across 3 lanes for samples
</gallery>


[[Category:ComboLock]] [[Category:20160922]]
[[Category:ComboLock]] [[Category:20160922]]

Latest revision as of 23:13, 29 September 2016

PCR Testing (Started Thursday)[edit]

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[edit]

  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 21 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 22 50 NTC
      Sample 1A 1.00E+09 H5 37 2 25 2* 2* 19 50 MM4
      Sample 1B 1.00E+09 H6 37 2 25 2* 2* 19 50 MM4
      Sample 1C 1.00E+09 H7 37 2 25 2* 2* 19 50 MM4
      NTC 0 H8 37 0 25 2* 2* 19 50 NTC
      Sample 1A 1.00E+09 C1 37 2 25 0.2* 0.2* 22.6 50 MM5
      Sample 1B 1.00E+09 C2 37 2 25 0.2* 0.2* 22.6 50 MM5
      Sample 1C 1.00E+09 C3 37 2 25 0.2* 0.2* 22.6 50 MM5
      NTC 0 C4 37 0 25 0.2* 0.2* 22.6 50 NTC
    3. Make master mixes according to following table
    4. Reagent Master Mix
      MM1 MM2 MM3 MM4 MM5
      PrimerF (10 uM) 15.5 3.1 1.55 6.2* 0.62*
      PrimerR (10 uM) 15.5 3.1 1.55 6.2* 0.62*
      KAPA Master Mix 77.5 77.5 77.5 77.5 77.5
      nfH2O 40.3 65.1 68.2 58.9 70.06
    5. Add 48 uL correct master mix to each well
    6. qPCR Cycles
      1. 95C 3 min
      2. 95C 3 sec
      3. 60C 30 sec
      4. 72C 20 sec
      5. plate read
      6. goto b x26
      7. 72C 2 min
      8. 16C hold

    Results[edit]