Hosuk:LabNotes/2014-5-30

From ZhangLabWiki
Jump to navigation Jump to search

Rolony from Mouse Brain Tissue Section : 4rd Try[edit]

  • Pepsin for 10min at 37C


Procedure[edit]

  • 05/28
    1. Attach the glass cover slip with a mounted tissue section to the Mattek dish using cyanoacrylate adhesive
    2. Wash twice using DEPC-treated H2O for 5 min each
    3. Add 0.25% Triton X-100 in DEPC-treated H2O for 15 min (S1, 10min for S2) and aspirate
    4. Wash with DEPC-treated H2O twice
    5. Add 200 ul 0.1% pepsin in 0.1 N HCl for 10 min at 37C
    6. Wash with 2 ml PBS three times to inactivate pepsin
    7. Add RT mix (Hexamer, -a-dUTP)
      • DEPC-H2O 161uL
      • M-MuLV Buffer 20uL
      • 25mM dNTP 2uL
      • 100uM random hexamer RT Primer 5uL
      • Rnase Inhibitor 2uL
      • M-MuLV reverse transcriptase 10 uL
    8. Incubate the specimen with the reaction mixture for 10 min at 4°C, then transfer to 37°C for overnight.


  • 05/29
    1. 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. 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 2hr.
      • 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.
    9. 1uL of 100uM RCA Primer + 199uL 2X SSC with 30% Formamide
    10. Aspirate and add 2XSSC, incubate at 60C for 10min
    11. Aspirate and Wash with 2X SSC, 1X SSC and PBS once each
    12. Prepare RCA reaction mix
      • H2O 168uL
      • Buffer 20uL
      • 25mM dNTP 4uL
      • 2mM a-dUTP 4uL
      • Phi29 4uL
    13. Add RCA mix, incubate at 30C for 17.5hr


  • 05/30
    1. 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
    10. Imaging


Result[edit]

  • almost all part of tissue were removed
  • But the remained regions has many rolonies.




File:TissueTest 4th 053014 20x Pos1.png File:TissueTest 4th 053014 20x Pos2.png


File:TissueTest 4th 053014 63x Pos1-Pos1.png File:TissueTest 4th 053014 63x Pos2-Pos1.png File:TissueTest 4th 053014 63x Pos2-Pos2.png


File:TissueTest 4th 053014 63x Pos1-Pos1 zoom4.png File:TissueTest 4th 053014 63x Pos2-Pos1 zoom4.png