Hosuk:LabNotes/2013-5-20
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Add circular DNA (~50pmole) in fixed cells and run RCA in the oven[edit]
- Produce circular DNA (50pmole input!) as same one made at 05/
- 20uL of CircLigase II product + 30uL 2x SSC in the fixed cell dish
- Run RCA in the oven (set temp: 33C) for 19 hour
- After RCA,
- Add 20 ul 250 mM BS(PEG)9 in 1 ml PBS for 1 hr at RT.
- Wash with PBS once
- Block with 1M Tris pH 8.0 for 30 min.
- Wash with PBS once
- Add 5 ul Riboshredder (Epicentre)+ 5ul RNase H(Enzymatics) in 190ul RNase H buffer(Enzymatics) for 1 hr at 37C.
- Rinse the entire well with ddH2O twice to remove traces of phosphate.
- Prepare 100uM of Cy3 labeled probe (Cy3-TCTTCAGCGTTCCCTAGA)
- 2uL of 100uM probes in 200uL 2x SSC
- Pre-heated probe solution between 60C and 80C for 5min
- Add to cells at room temp, and let it cool down over 5min
- Wash with 2x SSC (2mL, but 200uL work too) twice and image!
Result[edit]
- RCA rolony signal were from Cy3 and FITC channel very well.
- There were a lot of big strong spots, which were seen every time before, and I don’t think that’s rolonies,
- they looked cluster or chuck of RCA products so the Cy3 adpaters were bound there.
- Anyway, FITC channel represents Rolonies well…
- Let’s try Confocal imaging!
- Let’s move on a real protocol, from RT to RCA!
20x, Position 2[edit]
40x Oil, Position 2[edit]
File:S4 40xOil pos2 052013.png