Hosuk:LabNotes/2015-2-5
Jump to navigation
Jump to search
Human Brain Tissue : CA12k_Nov2014 V4 & V7 : 1st Try[edit]
3rd day[edit]
- Continued from 02/04
- 02/05
- For V4 & V7 samples: RCA Primer, final concentration : 0.5uM in 200uL (2x SSC) at 60C for 1hr.
- 1uL of 100uM RCA Primer + 199uL 2X SSC with 30% Formamide
- Wash with 2XSSC twice, and 1X SSC twice
- Prepare RCA reaction mix
- H2O 172uL
- Buffer 20uL
- 25mM dNTP 2uL
- 2mM a-dUTP 4uL
- Phi29(low conc.) 2uL
- Add RCA mix, incubate at 30C overnight
- For 1st Rolony generation : Wash with PBS once
- Prepare 4uL of BS(PEG)9 in 196uL PBS
- add 200uL BS(PEG)9 mix, incubate for 1hr at RT
- Wash with PBS once, add Tris pH 8.0, incubate for 30min
- Wash with PBS once
- Prepare detection Probe : Final Conc. 1uM --> 2uL of 100uM Cy3-Adapt + 198uL (2xSSC + 30% Formamide)
- Preheat detection Probe at 80C for 5min
- Add to sample and incubate at RT for 10min
- Wash two times for 1 minute each with 1 mL 1X PBS