Hosuk:LabNotes/2015-2-5

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Human Brain Tissue : CA12k_Nov2014 V4 & V7 : 1st Try[edit]

3rd day[edit]


  • 02/05
    1. For V4 & V7 samples: RCA Primer, final concentration : 0.5uM in 200uL (2x SSC) at 60C for 1hr.
    2. 1uL of 100uM RCA Primer + 199uL 2X SSC with 30% Formamide
    3. Wash with 2XSSC twice, and 1X SSC twice
    4. Prepare RCA reaction mix
      • H2O 172uL
      • Buffer 20uL
      • 25mM dNTP 2uL
      • 2mM a-dUTP 4uL
      • Phi29(low conc.) 2uL
    5. Add RCA mix, incubate at 30C overnight


    1. For 1st Rolony generation : Wash with PBS once
    2. Prepare 4uL of BS(PEG)9 in 196uL PBS
    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. 1uM --> 2uL of 100uM Cy3-Adapt + 198uL (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