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

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>Djacobse
>Djacobse
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| width="65" | 2X Kapa SYBR qPCR MM
| width="65" | 2X Kapa SYBR qPCR MM
| width="65" | 100 uM AmpF 6.4
| width="65" | 100 uM AmpF 6.4
| width="65" | 10 uM AmpR-IndXX
| width="65" | 10 uM AmpR6.3-IndXX
| width="65" | H2O
| width="65" | H2O
| width="65" | Total Volume (uL)
| width="65" | Total Volume (uL)
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| align="center" valign="bottom" | A1
| align="center" valign="bottom" | A1
| align="center" valign="bottom" | B1
| align="center" valign="bottom" | B1
| align="center" align="center" valign="bottom" | 71
| align="center" align="center" valign="bottom" | 1
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| 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" valign="bottom" | A2
| align="center" valign="bottom" | A2
| align="center" valign="bottom" | B2
| align="center" valign="bottom" | B2
| align="center" align="center" valign="bottom" | 72
| align="center" align="center" valign="bottom" | 2
| align="center" align="center" valign="bottom" | 2
| align="center" align="center" valign="bottom" | 2
| align="center" align="center" valign="bottom" | 25
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| align="center" valign="bottom" | A3
| align="center" valign="bottom" | A3
| align="center" valign="bottom" | B3
| align="center" valign="bottom" | B3
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| align="center" align="center" valign="bottom" | 3
| align="center" align="center" valign="bottom" | 2
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| align="center" valign="bottom" | A4
| align="center" valign="bottom" | A4
| align="center" valign="bottom" | B4
| align="center" valign="bottom" | B4
| align="center" align="center" valign="bottom" | 74
| align="center" align="center" valign="bottom" | 4
| align="center" align="center" valign="bottom" | 2
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| align="center" align="center" valign="bottom" | 25
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| align="center" valign="bottom" | A5
| align="center" valign="bottom" | A5
| align="center" valign="bottom" | B5
| align="center" valign="bottom" | B5
| align="center" align="center" valign="bottom" | 75
| align="center" align="center" valign="bottom" | 5
| align="center" align="center" valign="bottom" | 2
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| align="center" align="center" valign="bottom" | 25
| align="center" align="center" valign="bottom" | 25
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| align="center" valign="bottom" | A6
| align="center" valign="bottom" | A6
| align="center" valign="bottom" | B6
| align="center" valign="bottom" | B6
| align="center" align="center" valign="bottom" | 76
| align="center" align="center" valign="bottom" | 6
| align="center" align="center" valign="bottom" | 2
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| align="center" align="center" valign="bottom" | 25
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| align="center" valign="bottom" | A7
| align="center" valign="bottom" | A7
| align="center" valign="bottom" | B7
| align="center" valign="bottom" | B7
| align="center" align="center" valign="bottom" | 77
| align="center" align="center" valign="bottom" | 7
| align="center" align="center" valign="bottom" | 2
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| align="center" valign="bottom" | A8
| align="center" valign="bottom" | A8
| align="center" valign="bottom" | B8
| align="center" valign="bottom" | B8
| align="center" align="center" valign="bottom" | 78
| align="center" align="center" valign="bottom" | 8
| align="center" align="center" valign="bottom" | 2
| align="center" align="center" valign="bottom" | 2
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| align="center" align="center" valign="bottom" | 25

Revision as of 16:41, 24 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 AmpR Index Cells 2X Kapa SYBR qPCR MM 100 uM AmpF 6.4 10 uM AmpR6.3-IndXX H2O Total Volume (uL)
      Sample 1 A1 B1 1 2 25 0.5 5 17.5 50
      Sample 2 A2 B2 2 2 25 0.5 5 17.5 50
      Sample 3 A3 B3 3 2 25 0.5 5 17.5 50
      Sample 4 A4 B4 4 2 25 0.5 5 17.5 50
      Sample 5 A5 B5 5 2 25 0.5 5 17.5 50
      Sample 6 A6 B6 6 2 25 0.5 5 17.5 50
      Sample 7 A7 B7 7 2 25 0.5 5 17.5 50
      Sample 8 A8 B8 8 2 25 0.5 5 17.5 50
    3. Master Mix
      1. 200 uL KAPA SyberFast MM
      2. 140 uL nfH2O
      3. 4 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