Daniel:Notebook/ComboLock/2016-8-19: Difference between revisions

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>Djacobse
>Djacobse
 
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<li>Incubate reaction at 37 ºC for 2 hours</li>
<li>Incubate reaction at 37 ºC for 2 hours</li>
<li>Heat inactivate enzyme by incubating at 94C for 5 minutes</li>
<li>Heat inactivate enzyme by incubating at 94C for 5 minutes</li>
</ol>
<li>Elution</li>
<ol type="A">
<li>Add 25 uL prewarmed elution buffer; vortex to suspend</li>
<li>Incubate at RT for 2 minutes</li>
<li>Apply magnet and transfer supernatant to a clean centrifuge tube</li>
<li>Repeat elution with 25 uL fresh elution buffer; add supernatant to first elution</li>
</ol>
</ol>
<li>qPCR</li>
<li>qPCR</li>
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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:#FCD5B4;font-size:12pt;font-weight:bold" align="center"
|- style="background-color:#FCD5B4;font-size:12pt;font-weight:bold" align="center"
| width="65" height="45" | Sample
| width="105" height="45" | Sample
| width="165" | Name
| width="65" | Lane
| width="65" | Lane
| width="65" | AmpR Index
| width="65" | AmpR Index
Line 41: Line 35:
|- style="font-size:12pt"
|- style="font-size:12pt"
| height="15"  valign="bottom" | Sample 1
| height="15"  valign="bottom" | Sample 1
|  valign="bottom" | Amp1 Test
| align="center" valign="bottom" | A1
| align="center" valign="bottom" | A1
| align="center" align="center" valign="bottom" | 10
| align="center" align="center" valign="bottom" | 10
Line 52: Line 47:
|- style="background-color:#D9D9D9;font-size:12pt"
|- style="background-color:#D9D9D9;font-size:12pt"
| height="15"  valign="bottom" | Sample 2
| height="15"  valign="bottom" | Sample 2
|  valign="bottom" | Amp3 Test
| align="center" valign="bottom" | B1
| align="center" valign="bottom" | B1
| align="center" align="center" valign="bottom" | 11
| align="center" align="center" valign="bottom" | 11
Line 63: Line 59:
|- style="font-size:12pt"
|- style="font-size:12pt"
| height="15"  valign="bottom" | Sample 3
| height="15"  valign="bottom" | Sample 3
|  valign="bottom" | Latch X2 Specificity
| align="center" valign="bottom" | C1
| align="center" valign="bottom" | C1
| align="center" align="center" valign="bottom" | 12
| align="center" align="center" valign="bottom" | 12
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| align="center" align="center" valign="bottom" | 50
| align="center" align="center" valign="bottom" | 50


|- style="font-size:12pt"
|- style="background-color:#D9D9D9;font-size:12pt"
|style="background-color:#D9D9D9" height="15"  valign="bottom" | Sample 4
| height="15"  valign="bottom" | Sample 4
|style="background-color:#D9D9D9" align="center" valign="bottom" | D1
| valign="bottom" | Latch X3 Specificity
| align="center" valign="bottom" | D1
| align="center" align="center" valign="bottom" | 13
| align="center" align="center" valign="bottom" | 13
|style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 2
| align="center" align="center" valign="bottom" | 2
|style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 25
| align="center" align="center" valign="bottom" | 25
|style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 0.5
| align="center" align="center" valign="bottom" | 0.5
|style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 5
| align="center" align="center" valign="bottom" | 5
|style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 17.5
| align="center" align="center" valign="bottom" | 17.5
|style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 50
| align="center" align="center" valign="bottom" | 50


|- style="font-size:12pt"
|- style="font-size:12pt"
| height="15"  valign="bottom" | Sample 5
| height="15"  valign="bottom" | Sample 5
|  valign="bottom" | No Template
| align="center" valign="bottom" | E1
| align="center" valign="bottom" | E1
|style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 14
| align="center" align="center" valign="bottom" | 14
| 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
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| align="center" align="center" valign="bottom" | 50
| align="center" align="center" valign="bottom" | 50


|- style="font-size:12pt"
|- style="background-color:#D9D9D9;font-size:12pt"
|style="background-color:#D9D9D9" height="15"  valign="bottom" | Sample 6
| height="15"  valign="bottom" | Sample 6
|style="background-color:#D9D9D9" align="center" valign="bottom" | F1
| valign="bottom" | No Latch
| align="center" valign="bottom" | F1
| align="center" align="center" valign="bottom" | 15
| align="center" align="center" valign="bottom" | 15
|style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 2
| align="center" align="center" valign="bottom" | 2
|style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 25
| align="center" align="center" valign="bottom" | 25
|style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 0.5
| align="center" align="center" valign="bottom" | 0.5
|style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 5
| align="center" align="center" valign="bottom" | 5
|style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 17.5
| align="center" align="center" valign="bottom" | 17.5
|style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 50
| align="center" align="center" valign="bottom" | 50


|- style="font-size:12pt"
|- style="font-size:12pt"
| height="15"  valign="bottom" | Sample 7
| height="15"  valign="bottom" | Sample 7
|  valign="bottom" | No Padlock
| align="center" valign="bottom" | G1
| align="center" valign="bottom" | G1
| align="center" align="center" valign="bottom" | 16
| align="center" align="center" valign="bottom" | 16
Line 118: Line 119:
|- style="background-color:#D9D9D9;font-size:12pt"
|- style="background-color:#D9D9D9;font-size:12pt"
| height="15"  valign="bottom" | Sample 8
| height="15"  valign="bottom" | Sample 8
|  valign="bottom" | PCAmp2 Only
| align="center" valign="bottom" | H1
| align="center" valign="bottom" | H1
| align="center" align="center" valign="bottom" | 17
| align="center" align="center" valign="bottom" | 17
Line 129: Line 131:
|- style="font-size:12pt"
|- style="font-size:12pt"
| height="15"  valign="bottom" | Sample 9
| height="15"  valign="bottom" | Sample 9
|  valign="bottom" | PCR NTC
| align="center" valign="bottom" | A12
| align="center" valign="bottom" | A12
| align="center" align="center" valign="bottom" | 18
| align="center" align="center" valign="bottom" | 18
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</ol>
</ol>
</ol>
</ol>
===Results===
<gallery perrow=2 heights=300px widths=300px>
File:20160819-qPCR-PosControlTest.png|qPCR Curve
File:2016-08-19-PosControl-qPCR.png|Gel Image
</gallery>
It looks like lane 3 and 4 have bands at the correct expected size (231bp). None of the negative controls (5-8) have it, and lane 1 might also have it. This would indicate lane 2 had an error in the process. I can size select the bands and get ready for library prep if that is the case.


[[Category:ComboLock]] [[Category:20160818]]
[[Category:ComboLock]] [[Category:20160818]]

Latest revision as of 20:25, 19 August 2016

Positive Control Amplicon Test (Started yesterday)[edit]

Back to Calendar

Protocol[edit]

  1. Circularization
    1. Heat inactivate enzyme by incubating for 2 minutes at 94C
  2. Exonuclease Digestion
    1. Prepare exonuclease I/III mix by mixing 15 uL exonuclease I (20 units/μL) and 3 uL exonuclease III (100 units/μL) in 1:1 ratio
    2. Add 2 μL of exonuclease I/III mix to reaction
    3. Mix the reaction by swirling pipette around the well 5 times
    4. Incubate reaction at 37 ºC for 2 hours
    5. Heat inactivate enzyme by incubating at 94C for 5 minutes
  3. qPCR
    1. Set up reaction according to table
    2. Sample Name Lane AmpR Index Sample Vol (uL) 2X Kapa SYBR qPCR MM 100 uM AmpF 6.4 10 uM AmpR6.3-IndXX H2O Total Volume (uL)
      Sample 1 Amp1 Test A1 10 2 25 0.5 5 17.5 50
      Sample 2 Amp3 Test B1 11 2 25 0.5 5 17.5 50
      Sample 3 Latch X2 Specificity C1 12 2 25 0.5 5 17.5 50
      Sample 4 Latch X3 Specificity D1 13 2 25 0.5 5 17.5 50
      Sample 5 No Template E1 14 2 25 0.5 5 17.5 50
      Sample 6 No Latch F1 15 2 25 0.5 5 17.5 50
      Sample 7 No Padlock G1 16 2 25 0.5 5 17.5 50
      Sample 8 PCAmp2 Only H1 17 2 25 0.5 5 17.5 50
      Sample 9 PCR NTC A12 18 0 25 0.5 5 19.5 50
    3. Master Mix
      1. 227.5 uL KAPA SyberFast MM
      2. 159.25 uL nfH2O
      3. 4.55 uL AmpF 6.4
    4. Add 43 uL 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 x20
      7. 72C 2 min
      8. 16C hold

    Results[edit]

    It looks like lane 3 and 4 have bands at the correct expected size (231bp). None of the negative controls (5-8) have it, and lane 1 might also have it. This would indicate lane 2 had an error in the process. I can size select the bands and get ready for library prep if that is the case.