Hosuk:LabNotes/2014-4-1

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Revision as of 18:17, 1 April 2014 by >Hosuki78 (Created page with "*LabNote ===Cancer Cell : new Padlock probes and Ampligase of new RT Primers sample=== * Continue from 03/31 *04/01 : Start R...")
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Cancer Cell : new Padlock probes and Ampligase of new RT Primers sample[edit]

  • Continue from 03/31
  • 04/01 : Start RCA (@30C for 20hr.)
  • RCA procedure
    1. 1ul of RCA primer(200 uM, GATATCGGGAAGCTGA*A*G) in 199ul of 2xSSC/30% formamide
    2. Preheat primer at 60C (Tm = 55C when not in 30% formamide)
    3. Add 200ul and incubate at 45C for 15min.
    4. Aspirate, and wash using 2x SSC twice
    5. Aspirate, and wash using 0.1x SSC twice


  • Prepare the rolling circle amplification reaction mixtures on ice.
    1. 171 ul ddH2O,
    2. 20 ul 10x Phi29 buffer,
    3. 2 ul 25 mM dNTP,
    4. 4 ul 2 mM aminoallyl dUTP
    5. 3 ul Phi29 DNA polymerase (Epicentre, low concentration)
  • 200ul Total Volume


  • Add 200ul RCA mix and incubate at 30C for 20hr.