Hosuk:LabNotes/2015-2-4

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

1st and 2nd day[edit]

  • Samples : BA8 on Vectabond treated cover glass mounted at 2014-12-14
    • There are only the BA8s on Vectabond treated left.


  • 3 BA8 prepared
    • S1 : V4 Padlock probe
    • S2 : V7 Padlock probe
    • S3 : 1st Rolony generation
  • CA12k_Nov2014 Padlock Probes
    • Made at 2015-01-15 by Matt
    • V4 : 1337nM (38ul) --> 7.5uL for 100nM in 100uL Ampligase Mix
    • V7 : 803nM (18ul) --> 12.5uL for 100nM in 100uL Ampligase Mix


Procedure[edit]

  • 02/03
    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.


  • 02/04
    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 24hr.
      • H2O 72.5uL for V4, 67.5uL for V7
      • Buffer 10uL
      • Padlock Probe 7.5uL for V4, 12.5 uL for V7
      • Ampligase 10uL
    6. For 1st Rolony generation : RCA Primer, final concentration : 0.5uM in 200uL (2x SSC + 30% Formamide) 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