Hosuk:LabNotes/2014-4-1
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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
- 1ul of RCA primer(200 uM, GATATCGGGAAGCTGA*A*G) in 199ul of 2xSSC/30% formamide
- Preheat primer at 60C (Tm = 55C when not in 30% formamide)
- Add 200ul and incubate at 45C for 15min.
- Aspirate, and wash using 2x SSC twice
- Aspirate, and wash using 0.1x SSC twice
- Prepare the rolling circle amplification reaction mixtures on ice.
- 171 ul ddH2O,
- 20 ul 10x Phi29 buffer,
- 2 ul 25 mM dNTP,
- 4 ul 2 mM aminoallyl dUTP
- 3 ul Phi29 DNA polymerase (Epicentre, low concentration)
- 200ul Total Volume
- Add 200ul RCA mix and incubate at 30C for 20hr.