Hosuk:LabNotes/2015-2-14

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Human Brain Tissue : CA12k_Nov2014 V4 & V7 : 2nd Try[edit]

Sample[edit]

  • Samples : BA8 on Vectabond treated cover glass mounted at 2014-12-14
  • 2 BA8 prepared using double-side tape!
    • S1 : V4 Padlock probes, S2 : 1st Rolonies
    • I think I used 0.1% Pepsin instead of 0.01% by mistake, so I did 0.01% Pepsin for sure!
    • S2 dish got leak but S1 is totally fine...


  • CA12k_Nov2014 Padlock Probes
    • Made at 2015-02-02 by Matt
    • V4 : 819nM (58ul) --> 11.8uL for 100nM in 100uL Ampligase Mix


Procedure[edit]

  • 02/12
    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 10~15min 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 10 min, Wash with PBS 3 times
    8. Add - Pepsin incubation 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.


  • 02/13
    1. Skip BS(PEG)9 step, Wash with PBS once
    2. Add RNase H mix, incubate for 1hr. at 37C
      • 2 ul Riboshredder (Epicentre)
      • 10 ul RNase H(Enzymatics)
      • 20 ul RNase H buffer(Enzymatics)
      • 168 uL H2O
    3. Wash with nucleaus-free H2O twice
    4. For 1st Rolony Sample:Add CircLigase II mix, incubate at 60C for 2hr.
      • H2O 128uL
      • Buffer 20uL
      • 50mM MnCl2 10uL
      • 5M Betaine 40uL
      • CircLigaseII 2uL
    5. For Ampligase Sample:Add Ampligase mix, incubate at 37C for 30min. then move to 60C for 6hr.
      • H2O 68.2uL for V4
      • Buffer 10uL
      • Padlock Probe 11.8uL for V4
      • Ampligase 10uL
    6. For Both S1(V4 PP) and S2(1st Rolony generation) : RCA Primer, final concentration : 0.5uM in 200uL (2x SSC) at 60C for 1hr.
    7. 1uL of 100uM RCA Primer + 199uL 2X SSC with 30% Formamide
    8. Wash with 2XSSC twice, and 1X SSC twice
    9. Prepare RCA reaction mix
      • H2O 172uL
      • Buffer 20uL
      • 25mM dNTP 2uL
      • 2mM a-dUTP 4uL
      • Phi29(low conc.) 2uL
    10. Add RCA mix, incubate at 30C overnight


  • 02/14
    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