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

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>Djacobse
>Djacobse
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{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
{| 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"
|- style="background-color:#B8CCE4;font-size:12pt;font-weight:bold" align="center"
| width="105" height="45" | Sample
| width="65" height="45" | Sample
| width="65" | Molecule Count
| width="65" | Molecule Count
| width="65" | Lane
| width="65" | Lane
Line 126: Line 126:
| 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
Line 178: Line 178:
| 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
Line 189: Line 189:
| 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
Line 202: Line 202:
| 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
Line 215: Line 215:
| 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
Line 228: Line 228:
| 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
Line 241: Line 241:
| 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
Line 254: Line 254:
| 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
Line 267: Line 267:
| 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
Line 289: Line 289:


<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="119" 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>

Revision as of 18:45, 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 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. 55C 30 sec
      4. 72C 20 sec
      5. plate read
      6. goto b x26
      7. 72C 2 min
      8. 16C hold