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

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|- 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="105" height="45" | Sample
| width="105" height="45" | Sample
| width="140" | Name
| width="65" | Lane
| width="65" | Lane
| width="65" | AmpR Index
| width="65" | AmpR Index
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|- 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
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|- 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
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|- 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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|- style="font-size:12pt"
|- style="font-size:12pt"
|style="background-color:#D9D9D9" height="15"  valign="bottom" | Sample 4
|style="background-color:#D9D9D9" height="15"  valign="bottom" | Sample 4
|style="background-color:#D9D9D9"  valign="bottom" | Latch X3 Specificity
|style="background-color:#D9D9D9" align="center" valign="bottom" | D1
|style="background-color:#D9D9D9" align="center" valign="bottom" | D1
| align="center" align="center" valign="bottom" | 13
| align="center" align="center" valign="bottom" | 13
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|- 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
|style="background-color:#D9D9D9" align="center" align="center" valign="bottom" | 14
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|- style="font-size:12pt"
|- style="font-size:12pt"
|style="background-color:#D9D9D9" height="15"  valign="bottom" | Sample 6
|style="background-color:#D9D9D9" height="15"  valign="bottom" | Sample 6
|style="background-color:#D9D9D9"  valign="bottom" | No Latch
|style="background-color:#D9D9D9" align="center" valign="bottom" | F1
|style="background-color:#D9D9D9" align="center" valign="bottom" | F1
| align="center" align="center" valign="bottom" | 15
| align="center" align="center" valign="bottom" | 15
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|- 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
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|- 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
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|- 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

Revision as of 16:16, 19 August 2016

Positive Control Amplicon Test (Started yesterday)

Back to Calendar

Protocol

  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. Elution
    1. Add 25 uL prewarmed elution buffer; vortex to suspend
    2. Incubate at RT for 2 minutes
    3. Apply magnet and transfer supernatant to a clean centrifuge tube
    4. Repeat elution with 25 uL fresh elution buffer; add supernatant to first elution
  4. 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