Daniel:Notebook/ComboLock/2016-7-6

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Revision as of 22:40, 6 July 2016 by >Djacobse
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Sequential Hybridization Test (Started yesterday)

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Protocol

  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 22 2 25 0.5 5 17.5 50
      Sample 2 A2 B2 23 2 25 0.5 5 17.5 50
      Sample 3 A3 B3 24 2 25 0.5 5 17.5 50
      Sample 4 A4 B4 25 2 25 0.5 5 17.5 50
      Sample 5 A5 B5 26 2 25 0.5 5 17.5 50
      Sample 6 A6 NTC 27 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

    Protocol Part II

    Since lanes 1-5 showed product, I'm going to sequence all of them. This is disheartening as MORE samples showed signal now that I changed to sequential hybridization.

    1. Size Selection
      1. Combine 20 uL sample from each of the five samples
        1. Add only 10 uL from lane 3 plus 10 uL TBE since lane 3 had greatest signal
      2. Add 30 uL 6X loading dye
      3. Aliquot 30 uL each into 4 lanes
      4. 2 uL ladder with 5 uL gel loading dye and 23 uL TBE buffer
      5. Run gel for 45 min at 250V
      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

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

    Continued from results on 7-1-2016

    Source ng/uL uL added final mass (ng)
    Ab1 15 20 300

    Sequencing Sample: DEJ-CL-P01

    C Probe SOD1/VIM Test (Started 6-29-2016)

    Continued from 7-1. I added more human than mouse since I am more interested in the human samples (although it is still important to check the mouse).

    Sample ng/uL uL added final mass (ng)
    Hs-Sample 2 38.5 15 577.5
    mm-Sample 4 43.3 10 433

    Sequencing Sample: DEJ-CL-C02