Hosuk:LabNotes/2014-7-21

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Cancer Cell : Ampligase and gap filling for Mutation detection of Cancer Cell[edit]

Samples[edit]

  • Two NCIH1975 made at 07/15 – both of them made with rHex
  • Two NCIH1975 made at 07/09 – one made with rHex, another one made with Top48 RT Primer


    • S1_0715 : +Ampligase and +KlenTaq at 45C for 24hr.
    • S2_0715 : +Ampligase and +KlenTaq at 60C for 24hr.
    • S1_0709_rHex : (No)-Ampligase and +KlenTaq at 45C for 24hr.
    • S2_0709_Top48 : +Ampligase and +KlenTaq at 45C for 24hr.


Procedure[edit]

  • 07/21 : Start Ampligase (~10:30am)
  • 07/22 : Start RCA
  • 07/23 : End RCA


  • 1ul of RCA primer(FISSEQ_ppRCA, 200 uM, GATATCGGGAAGCTGA*A*G) in 199ul of 2xSSC
  • Preheat primer at 60C (Tm = 55C when not in 30% formamide)
  • Add 200ul and incubate at 45C for 30min.
  • Aspirate, and wash using 2x SSC twice
  • Aspirate, and wash using 0.1x SSC twice
  • Prepare the rolling circle amplification reaction mixtures on ice.
    • 172 ul ddH2O,
    • 20 ul 10x Phi29 buffer,
    • 2 ul 25 mM dNTP,
    • 4 ul 2 mM aminoallyl dUTP
    • 2 ul Phi29 DNA polymerase (Epicentre, low concentration)
    • 200ul Total Volume


  • Finished All process at 07/23