Hosuk:LabNotes/2014-12-12

From ZhangLabWiki
Jump to navigation Jump to search

Human Brain Tissue : mild Pepsin condition test[edit]

  • Samples from Yun
    • S1: BA8 --> the same size as before (larger one) but cut half with Razer
    • S2: BA22 --> smaller than BA8


Procedure[edit]

  • 12/10
    1. Take Tissue glass slide from -80C, and put it in a well of sterilized 6 well plate to 50C (hot plate) for 3min. (To prevent RNase activity)
    2. Add 4% formamide on the glass, incubate for 30min at room temp.
    3. Wash tissue with 2x SSPE twice for 5min each
    4. Aspirate 2x SSPE, and wipe solution around the edge of the glass
    5. Apply adhesive to glass, place glass to MatTek dish, wait for 20min for complete dry
    6. Wash with 2x SSPE twice (add DEPC-PBS on a well, aspirate)
    7. Add 0.25% Triton X-100 in 2x SSPE for 15 min
    8. Pepsin incubation - S2:none, S3:0.1%, S4:0.01% for 5 min each
    9. Wash with DEPC-H2O three times
    10. 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
    11. Incubate the specimen with the reaction mixture for 10 min at 4°C, then transfer to 37°C for overnight.


  • 12/11
    1. Skip BS(PEG)9 step, Wash with PBS once
    2. 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
    3. Wash with nucleaus-free H2O twice
    4. Add CircLigase II mix, incubate at 60C for 2hr.
      • H2O 128uL
      • Buffer 20uL
      • 50mM MnCl2 10uL
      • 5M Betaine 40uL
      • CircLigaseII 2uL
    5. RCA Primer, final concentration : 0.5uM in 200uL (2x SSC + 30% Formamide) at 60C for 1hr.
    6. 1uL of 100uM RCA Primer + 199uL 2X SSC with 30% Formamide
    7. Wash with 2XSSC twice, and 1X SSC twice
    8. Prepare RCA reaction mix
      • H2O 172uL
      • Buffer 20uL
      • 25mM dNTP 2uL
      • 2mM a-dUTP 4uL
      • Phi29(low conc.) 2uL
    9. Add RCA mix, incubate at 30C overnight


  • 12/12
    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


Result[edit]

  • Imaging condition
    • 20x, 4k, Pinhole = 4, Laser = 4, PMT = 620 (580 for S4), 20x 4K
  • Result : Quit good!


  • I don’t know why the rolonies are quit many for this time, but it maybe SSPE? Or less pepsin!
    • 0.1% Pepsin for 5 min is the best!
    • Need more detail scan with 63x obj., and DAPI or DRAQ5 staining and take images.


Result Images : prelim scanning[edit]

  • 20x, 4k