Hosuk:LabNotes/2014-8-7

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Revision as of 23:05, 12 August 2014 by >Hosuki78 (Created page with "*LabNote ===Cancer Mutation detection=== ====Sample==== *Sample **NCIH1975_S1_2014-08-05 : -dUTP, rHex  45C **NCIH1975_S2_2014-08-03 : +dUTP, rHex  ...")
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Cancer Mutation detection[edit]

Sample[edit]

  • Sample
    • NCIH1975_S1_2014-08-05 : -dUTP, rHex  45C
    • NCIH1975_S2_2014-08-03 : +dUTP, rHex  60C --> will use this later!


Run Ampligase + Klentaq and 2nd RCA[edit]

  • Reaction Mix

File:Ampligase Reagent 2014-08-07.jpg


  • Start Ampligase+Klentaq : 08/05, 3:30pm (Just use NCIH1975_S1_2014-08-05), ~21.5hr at 45C
  • Start RCA : 08/06, 2:40pm
    • 1uM ppRCA in 200uL 2x SSC, add and incubate at 42C for 36min,
    • Wash with 2x SSC twice, 1x SSC twice, 1x PBS twice, then add Phi29 mix
  • Finish : 08/07 (RCA for ~18.5hr)


Decoding[edit]

  • Decoding – 08/07
  • Sample : NCIH1975_S1_2014-08-05 : -dUTP, rHex
  • 1st Rolony – Cy3 : 1uM
  • dcProbe0~4 : 488, Cy3 : 1uM
    • dcProbe[#]-488 and dcProbe[#]-Cy3 are mixed in a sampe 100uL tube


  • Step
    1. Add dcProbe[#], incubate for 10min, image
    2. Strip with 80% formamide(pre-heated at 70C) for 15min, wash with PBS twice
    3. Repeat applying dcProbe and imaging


  • Result : under anaylzinging