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