Hosuk:LabNotes/2014-5-7

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Test : +/- a-dUTP, and long RT incubation at 4C[edit]


Procedure[edit]

  • 05/06
    1. 10% Formalin for 15min at RT(25C) --> done at CERC
    2. Wash with PBS 3 times --> done at CERC
    3. Move samples to the lab at PFBH
    4. Add 0.25% Triton X-100 in DEPC-PBS and incubate for 10min at room temp.
    5. mean while prepare RT mix and put the mix on an ice
    6. Aspirate 0.25% Triton X-100 in DEPC-PBS and wash with PBS 3 times
    7. Add RT mix and put in 4C, incubate for 15min. (1hr. for other 2 sample of target specific primers)
    8. Move samples to 37C and incubate for 12hr.


  • 05/07
    1. Wash with PBS once
    2. Prepare 18uL of BS(PEG)9 in 782uL PBS (for 4 dishes, volume per each dish is 200uL)
    3. add 200uL BS(PEG)9 mix, incubate for 1hr at RT
    4. Wash with PBS once, add Tris pH 8.0, incubate for 30min
    5. Add RNase H mix, incubate for 1hr. at 37C
      • RNase H mix : 2 ul Riboshredder (Epicentre)+ 10 ul RNase H(Enzymatics) in 20 ul RNase H buffer(Enzymatics) + 168 uL H2O
    6. Wash with nucleaus-free H2O twice
    7. Add CircLigase II mix, incubate at 60C for 3hr.
      • H2O 128uL
      • Buffer 20uL
      • 50mM MnCl2 10uL
      • 5M Betaine 40uL
      • CircLigaseII 2uL
    8. RCA Primer, final concentration : 0.5uM in 200uL (2x SSC + 30% Formamide) at 60C for 1hr.
      • 1uL of 100uM RCA Primer + 199uL 2X SSC with 30% Formamide
    9. Aspirate and add 2XSSC, incubate at 60C for 10min
    10. Aspirate and Wash with 2X SSC, 1X SSC and PBS once each
    11. Prepare RCA reaction mix
      • H2O 168uL
      • Buffer 20uL
      • 25mM dNTP 4uL
      • 2mM a-dUTP 4uL
      • Phi29 4uL
    12. Add RCA mix, incubate at 30C for 20hr


  • 05/08
    1. Wash with PBS once
    2. Prepare 18uL of BS(PEG)9 in 782uL PBS (for 4 dishes, volume per each dish is 200uL)
    3. add 200uL BS(PEG)9 mix, incubate for 1hr at RT
    4. Wash with PBS once, add Tris pH 8.0, incubate for 30min
    5. Wash with PBS once
    6. Prepare detection Probe : Final Conc. 2.5uM --> 5uL of 100uM Cy3-Adapt in 200uL (2xSSC + 30% Formamide)
    7. Preheat detection Probe at 80C for 5min
    8. Add to sample and incubate at RT for 10min
    9. Wash two times for 1 minute each with 1 mL 1X PBS
    10. Imaging