Hosuk:LabNotes/2013-4-24
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RCA with Pre-circularized DNA on fixed cells[edit]
Procedure[edit]
- Run 18hr. RCA with using CircLigase II product got from 4/24 result
- 7uL of product + 93uL 2x SSC in the fixed cell dish
- incubate 15min at RT
- Wash with PBS once
- Run RCA with the same step as rolony protocol
- 1uL of 100uM RCA primer in 199uL 2x SSC
- Add to cell, incubate for 15min at 60C
- Aspirate, and wash using 2x SSC twice
- Aspirate, and wash using 0.1x SSC twice
- Add RCA mix
- 18hr in 30C Oven
Result[edit]
- FL spots looked similar number and similar density to what I’ve got before (e.g. 03/05).
- Those seemed rolony but not many.
- It is suspected that rolonies didn't bind inside cell so they could be washed away.
- Are there problem in washing step? or BS(PEG)9 procedure?
- Need to ask to Jay