Daniel:Notebook/ComboLock/2016-7-26

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Latch 100 and Dextran Test (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 AmpR Index Cells 2X Kapa SYBR qPCR MM 100 uM AmpF 6.4 10 uM AmpR6.3-IndXX H2O Total Volume (uL)
      Sample 1 (Hs-Control) A1 9 2 25 0.5 5 17.5 50
      Sample 2 (Hs-Dextran) B1 10 2 25 0.5 5 17.5 50
      Sample 3 (Hs-Latch 100X) C1 11 2 25 0.5 5 17.5 50
      Sample 4 (Hs-Neg. Control) D1 12 2 25 0.5 5 17.5 50
      Sample 5 (mm-Control) E1 13 2 25 0.5 5 17.5 50
      Sample 6 (NTC) F1 14 2 25 0.5 5 17.5 50
      Sample 7 (No sample) G1 15 0 25 0.5 5 19.5 50
    3. Master Mix
      1. 177.5 uL KAPA SyberFast MM
      2. 124.25 uL nfH2O
      3. 3.55 uL AmpF 6.4 100 uM
    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]

    Library Prep[edit]

    1. Size Selection
      1. Combine 20 uL sample from lanes 1-5
      2. Add 20 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 25 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