Hosuk:LabNotes/2014-8-7
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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
- Add dcProbe[#], incubate for 10min, image
- Strip with 80% formamide(pre-heated at 70C) for 15min, wash with PBS twice
- Repeat applying dcProbe and imaging
- Result : under anaylzinging