Daniel:Notebook/ComboLock/2016-6-30: Difference between revisions

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>Djacobse
>Djacobse
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<ol type="a">
<ol type="a">
<li>165 uL KAPA SyberFast MM</li>
<li>165 uL KAPA SyberFast MM</li>
<li>115.5 uL nfH2O</li>
<li>116 uL nfH2O</li>
<li>3.3 uL AmpF 6.4</li></ol>
<li>3.3 uL AmpF 6.4</li></ol>
<li>Add 43 uL Master Mix to each well</li>
<li>Add 43 uL Master Mix to each well</li>

Revision as of 19:32, 30 June 2016

C Probe SOD1/VIM 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 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 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 9 2 25 0.5 5 17.5 50
      Sample 2 A2 B2 10 2 25 0.5 5 17.5 50
      Sample 3 A3 B3 11 2 25 0.5 5 17.5 50
      Sample 4 A4 B4 12 2 25 0.5 5 17.5 50
      Sample 5 A5 B5 13 2 25 0.5 5 17.5 50
      Sample 6 A6 NTC 14 2 25 0.5 5 19.5 50
    3. Master Mix
      1. 165 uL KAPA SyberFast MM
      2. 116 uL nfH2O
      3. 3.3 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

    Antibody Stage 2 Test 2 (Started 6-28-2016)

    1. Size Selection
      1. Take 20 uL sample from B2,B3,B5, and B6 into separate tubes
      2. Add 4 uL 6X loading dye to each tube
      3. Aliquot 24 uL each into 4 lanes (last one will have 20 uL)
      4. 5 uL ladder with 1 uL gel loading dye
      5. Run gel for 25 min at 220V
      6. Stain with 3 uL SYBR Gold for 3 min
      7. Image in gel doc; cut out selected bands and place in 0.5 mL tube with hole in the bottom inside a 1.5 mL tube
      8. Centrifuge gel at 14000rpm for 1.5 minutes
      9. Remove 0.5 mL tube and add 500 uL TE buffer to shredded gel
      10. Incubate at 37C for at least an hour with vigorous shaking
      11. Centrifuge at 14000rpm for 1.5 minutes
      12. Transfer supernatant to a nanosep column
      13. Centrifuge at 14000rpm for 1.5 minutes
      14. Transfer flow through to a 2 mL tube
    2. Ethanol Precipitation
      1. Add 1.25 mL 100% EtOH, 50 uL 3M NaOAc, and 2 uL glycoblue to sample
      2. Incubate overnight at -80C; continued tomorrow