Hosuk:LabNotes/2013-4-22
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Next Plan
- Run RCA reaction in a tube with CircLigase II reaction sample using custom DNA
- Since the custom DNA has the sequence for binding to RCA primer,
- I could see the signal from RCA product.
- Experiment
- Run CircLigase II with custom ssDNA (2hr. @ 60C)
- Run Exo I (1hr. @ 37C)
- Tube1 : custom ssDNA + RCA (18hr. @ 30C)
- Tube2 : Circ + RCA (18hr. @ 30C)
- Tube3 : Circ and Exo I + RCA (18hr. @ 30C)
- (As following Jay's suggestion, 18hour RCA was done from yesterday to today using fixed cells, but couldn't get any reasonable result. There were some FL signal, but it was false signal)
- Jay's suggestion
- for now, just wash the cells well before you fix. (2 ml PBS x 3)
- after fixing cells and treating with etoh, wash with PBS.
- then just dilute your DNA in 200 ul of 2x SSC and incubate at RT for 15 min.
- wash with 2xSSC several times.
- then just do your RCA. (with aminoallyl dUTP), followed by the BS(PEG)9 step. then hybridize. You should get a lot of rolonies.
- Jay's suggestion