Hosuk:LabNotes/2014-9-19

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ppCOL1A1 test – in NCIH1975[edit]

  • To know where the 2nd Rolonies of ppCOL1A1 present in a different cells (NCIH1975)


Sample[edit]

  • S1 : NCIH1975_RCA2014-08-02 : 1st Rolony : good
  • S2 : NCIH1975_RCA2014-08-05 : 1st Rolony : less than S1, but not bad


Procedure[edit]


  • Add 100ul Padlock Probe Mix
Components Volume
5 U/ul Ampligase 10
10X Ampligase Buffer 10
100uM ppCOL1A1 2
H2O 78
Total 100
  • Incubate at 37C for 30min
  • Incubate at 60C for 24hrs
  • Wash with 1X PBS twice
  • Add 200ul 0.5uM FISSEQ_ppRCA in 2X SSC + 30% formamide (pre-heated to 75C) and incubate at 45C for 30min
  • Wash with 2X SSC twice
  • Add 200ul RCA Mix and incubate at 30C for 16hrs
    • H2O : 172uL
    • 10x Buffer : 20uL
    • dNTP : 2uL
    • a-dUTP : 4uL
    • Phi29 (1000 U/µl) : 0.5uL
  • Wash with 1X PBS once
  • Add 200ul BS(PEG)9 (4ul BS(PEG)9 + 196ul 1X PBS) at RT for 1hr
  • Wash with 1X PBS twice
  • Add 200ul 1M Tris pH 8.0 at RT for 30min
  • Wash with 1X PBS twice and store in PBS



Result[edit]

  • In Sample1 (has more 1st and 2nd rolonies) --> Most of 2nd rolonies from ppCOL1A1 were found in nuclear.
  • In Sample2 (has less 1st and 2nd rolonies) --> Fewer 1st rolonies an 2nd rolonies compare to S1 has, but some of 2nd rolonies from ppCOL1A1 were found outside of nuclear.


File:LabnoteImg Comp MIP NCIH1975 ppCOL1A1Test S1 RCA20140802 1st 2nd BF 63x 2k Pos1.png


File:LabnoteImg Comp MIP NCIH1975 ppCOL1A1Test S2 RCA20140805 1st 2nd BF 63x 2k Pos2.png