Daniel:Notebook/ComboLock/2016-6-30

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

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Protocol[edit]

  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 10 2 25 0.5 5 17.5 50
      Sample 2 A2 B2 11 2 25 0.5 5 17.5 50
      Sample 3 A3 B3 12 2 25 0.5 5 17.5 50
      Sample 4 A4 B4 13 2 25 0.5 5 17.5 50
      Sample 5 A5 B5 14 2 25 0.5 5 17.5 50
      Sample 6 A6 NTC 15 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]

    It looks like the right number of samples amplified (One, kinda 2, but it was the wrong lanes. There is a possibility I confused the tubes at some point, so I'll repeat the experiment to see if that is true.

    Antibody Stage 2 Test 2 (Started 6-28-2016)[edit]

    I'm going to get libraries from each of the tests that I have done so far. Since the 6/28 experiment was the most recent antibody test, I'll be using those samples.

    1. Size Selection
      1. Combine 20 uL sample from each of the seven samples
      2. Add 30 uL 6X loading dye
      3. Aliquot 42 uL each into 4 lanes
      4. 4 uL ladder with 7 uL gel loading dye and 31 uL TBE buffer
      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

    C Probe Stage 2 Test Library Prep (From 6-25-2016)[edit]

    Finishing the library prep from the first C probe test. Just combining the samples to be equimolar (since they are the same size I'm assuming equimolar~equimass)

    Batch Source ng/uL uL added final mass (ng)
    Batch2 43.1 1.3 55.5
    Batch3 11.1 5 55.5
    Batch5 36.1 1.5 55.5
    Batch6 18.3 3.0 55.5