Daniel:Notebook/ComboLock/2016-7-2

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C Probe SOD1/VIM Test 2 (Started yesterday)[edit]

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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 10 uL exonuclease I (20 units/μL) and 2 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 16 2 25 0.5 5 17.5 50
      Sample 2 A2 B2 17 2 25 0.5 5 17.5 50
      Sample 3 A3 B3 18 2 25 0.5 5 17.5 50
      Sample 4 A4 B4 19 2 25 0.5 5 17.5 50
      Sample 5 A5 B5 20 2 25 0.5 5 17.5 50
      Sample 6 A6 NTC 21 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

    qPCR Results[edit]

    Human Cell Line Preparation[edit]

    The U87MG cells are ready to passage and I'm almost out of frozen cells.

    1. Fix cells
      1. Pellet cells at 600g for 3 min and resuspend in 5 mL medium with 1.6% paraformaldehyde
      2. Incubate 10 min at RT
      3. Wash cells with 5 mL wash buffer
      4. Pellet cells and permeabilize with ice-cold methanol for 10 min on ice
      5. Split cells into 1 mL fractions
      6. Once in methanol cells may be stored long term