Daniel:Notebook/ComboLock/2016-6-24: Difference between revisions

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>Djacobse
>Djacobse
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<ol type="A">
<ol type="A">
<li>Set up reaction according to table</li>
<li>Set up reaction according to table</li>
{| 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"
| width="134" height="45" | Sample
| width="65" | Lane
| width="65" | Batch
| width="65" | Cells
| width="65" | 2X Kapa SYBR qPCR MM
| width="65" | 100 uM Primer2
| width="65" | 100 uM Primer4-RC
| width="65" | H2O
| width="65" | Total Volume (uL)
|- style="font-size:12pt"
| height="15"  valign="bottom" | Sample 1
| align="center" valign="bottom" | A1
| align="center" valign="bottom" | B1
| align="center" align="center" valign="bottom" | 1
| align="center" align="center" valign="bottom" | 25
| align="center" align="center" valign="bottom" | 0.5
| align="center" align="center" valign="bottom" | 0.5
| align="center" align="center" valign="bottom" | 22
| align="center" align="center" valign="bottom" | 50
|- style="background-color:#D9D9D9;font-size:12pt"
| height="15"  valign="bottom" | Sample 2
| align="center" valign="bottom" | A2
| align="center" valign="bottom" | B2
| align="center" align="center" valign="bottom" | 1
| align="center" align="center" valign="bottom" | 25
| align="center" align="center" valign="bottom" | 0.5
| align="center" align="center" valign="bottom" | 0.5
| align="center" align="center" valign="bottom" | 22
| align="center" align="center" valign="bottom" | 50
|- style="font-size:12pt"
| height="15"  valign="bottom" | Sample 3
| align="center" valign="bottom" | A3
| align="center" valign="bottom" | B3
| align="center" align="center" valign="bottom" | 1
| align="center" align="center" valign="bottom" | 25
| align="center" align="center" valign="bottom" | 0.5
| align="center" align="center" valign="bottom" | 0.5
| align="center" align="center" valign="bottom" | 22
| align="center" align="center" valign="bottom" | 50
|- style="background-color:#D9D9D9;font-size:12pt"
| height="15"  valign="bottom" | Sample 4 (No Probe)
| align="center" valign="bottom" | A4
| align="center" valign="bottom" | B4
| align="center" align="center" valign="bottom" | 1
| align="center" align="center" valign="bottom" | 25
| align="center" align="center" valign="bottom" | 0.5
| align="center" align="center" valign="bottom" | 0.5
| align="center" align="center" valign="bottom" | 22
| align="center" align="center" valign="bottom" | 50
|}
<li>qPCR Cycles</li>
<li>qPCR Cycles</li>
<ol type="a">
<ol type="a">

Revision as of 23:54, 22 June 2016

Stage 2 Test (Started yesterday)

Back to Calendar

Protocol

  1. Latch and Padlock Hybridization
    1. Heat inactivate enzyme by incubating for 2 minutes at 94C
  2. Exonuclease Digestion
    1. Prepare exonuclease I/III mix by mixing 2 uL exonuclease I (20 units/μL) and 10 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 Lane Batch Cells 2X Kapa SYBR qPCR MM 100 uM Primer2 100 uM Primer4-RC H2O Total Volume (uL)
      Sample 1 A1 B1 1 25 0.5 0.5 22 50
      Sample 2 A2 B2 1 25 0.5 0.5 22 50
      Sample 3 A3 B3 1 25 0.5 0.5 22 50
      Sample 4 (No Probe) A4 B4 1 25 0.5 0.5 22 50
    3. 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