Daniel:Notebook/ComboLock/2016-6-28

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Antibody Stage 2 Test (Started yesterday)[edit]

Back to Calendar

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 2 uL exonuclease I (20 units/μL) and 10 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 1 2 25 0.5 5 17.5 50
      Sample 2 A2 B2 2 2 25 0.5 5 17.5 50
      Sample 3 A3 B3 3 2 25 0.5 5 17.5 50
      Sample 4 A4 B4 4 2 25 0.5 5 17.5 50
      Sample 5 A5 B5 5 2 25 0.5 5 17.5 50
      Sample 6 A6 B6 6 2 25 0.5 5 17.5 50
    3. Master Mix
      1. 165 uL KAPA SyberFast MM
      2. 115.5 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]

    File:VIM-Ab-LPTest Results 20160628.png

    qPCR Repeat[edit]

    1. 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 1 2 25 0.5 5 17.5 50
        Sample 2 A2 B2 2 2 25 0.5 5 17.5 50
        Sample 3 A3 B3 3 2 25 0.5 5 17.5 50
        Sample 4 A4 B4 4 2 25 0.5 5 17.5 50
        Sample 5 A5 B5 5 2 25 0.5 5 17.5 50
        Sample 6 A6 B6 6 2 25 0.5 5 17.5 50
        Sample 7 A7 NA 7 0 25 0.5 5 19.5 50
      3. Master Mix
        1. 192.5 uL KAPA SyberFast MM
        2. 134.75 uL nfH2O
        3. 3.85 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

      Results[edit]

      The second qPCR confirmed that I am seeing a good deal of signal in many of the lanes that shouldn't have any.

      Trial 2[edit]

      After talking with Andrew, I want to try yesterday's experiment again with a fixed number of cells (10000). This is to prevent any clogging that may occur during the protocol if there are too many cells.

      1. Hybridization
        1. Measure cell concentration using 10 uL cell sample in 90 uL nfH2O
          1. Human-3.9E5 cells/mL; Mouse-1.2E6 cells/mL
        2. Extract cells to a new tube such that there are 100000 cells present
          1. Human-256 uL; Mouse-85 uL
        3. Pellet cells via centrifugation at 600g for 3 min
        4. Add 200 uL Wash Buffer (0.05% Tween 20 in 1X PBS)
        5. Pellet cells via centrifugation at 600g for 3 min
        6. Add PLA mix and probes to create hybridization solution
        7.   uL PA Mix uL PB Mix uL PLA buffer
          PLA Mix E 1 1 4
          PLA Mix NC 0 0 6
        8. Resuspend cells in 10 uL PBS
        9. Mix 1 uL cell lysate with 3 uL Probe mix (10000 cells/reaction)
        10.   uL PLA Mix E uL PLA Mix NC uL U87MG Cells uL 3T3 Cells
          Sample 1 3 0 1 0
          Sample 2 3 0 0 1
          Sample 3 0 3 1 0
          Sample 4 0 3 0 1
          Sample 5 3 1 0 0
        11. Incubate 1.5 hours at RT
        12. Add 200 uL 0.05% Tween 20 in PBS
        13. Wash twice with 0.05% Tween 20 in PBS
          1. Pellet cells at 600xg for 3 min
      2. Latch and Padlock Hybridization
        1. Resuspend pellet in 100 uL LPH buffer per sample (600 uL S1, 200 uL S2)
        2. Add 1 uL of each insert/backbone (final conc. 100 nM each)
        3. Batch Cell Sample Latch0001 (1 uL) Padlock0001-68 (1 uL) Padlock0001-88 (1 uL)
          Batch 1 S1 X X  
          Batch 2 S1 X   X
          Batch 3 S2 X X  
          Batch 4 S2 X   X
          Batch 5 S3 X X  
          Batch 6 S4 X X  
          Batch 7 S5 X X  
        4. Incubate for 30 min at 37C following the sample matrix
        5. Wash twice with 200 uL wash buffer
          1. Wash by pelleting cells at 600xg for 3 min
        6. Resuspend with 20 uL 1x Amp Ligase buffer
      3. Circularization
        1. Prepare 20 uL KLN mix
        2. Add 2 uL KLN mix to 20 uL of reaction
        3. Incubate at 55C overnight (16 hours)
        4. Continued tomorrow

        Buffers[edit]

        1. Latch-Padlock Hybridization (LPH) Buffer
          1. 100 nM insert/backbone oligos
          2. 1X SSC
          3. 40 U/mL RNasin
          4. PBS
        2. KLN Mix 20 uL total
          1. 4 uL HemoKlentaq (20% v/v)
          2. 2 μL Ampligase (0.5 U/uL)
          3. 2 uL 1 mM dNTP mix (100 μM ea)
            1. Add 10 uL 10mM dNTP mix to 90 uL nfH2O
          4. 2 uL 1x Ampligase Buffer 10x
          5. 10 uL nfH2O

        LPH Buffer

        Reagent Stock Final Dilution Amt in 100 uL (uL)
        Oligos 10 uM 100 nM 100 1 uL
        SSC 20X 1X 5 20 uL
        Rnasin 40000 U/mL 40 U/mL 100 1 uL
        PBS 10X 1X 10 10 uL
        nf H2O NA NA NA 68 uL