Hosuk:LabNotes/2013-4-22: Difference between revisions
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*(As | *(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 | **Jay's suggestion | ||
***for now, just wash the cells well before you fix. (2 ml PBS x 3) | ***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. | ***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. | ***then just dilute your DNA in 200 ul of 2x SSC and incubate at RT for 15 min. | ||
***wash with 2xSSC several times. | ***wash with 2xSSC several times. | ||
***then just do your RCA. (with aminoallyl dUTP), followed by the | ***then just do your RCA. (with aminoallyl dUTP), followed by the BS(PEG)9 step. then hybridize. You should get a lot of rolonies. |
Revision as of 21:43, 22 April 2013
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.
- (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