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Hosuk:LabNotes/2013-4-24
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====RCA with Pre-circularized DNA on fixed cells==== =====Procedure===== *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===== *FL spots looked similar number and similar density to what Iβve got before (e.g. [[Hosuk:LabNotes/2013-3-5|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 {| {{table}} | 20x Obj. Bright field || | 20x Obj. Cy3 || | Merged || |- |([[Media:S1_washPBS3times_PreCirc_RCA18hr_Cy3Adapt_20x_pos02_fig04_BF_Exp0.2-EM00.tif|Original]]) || |([[Media:S1_washPBS3times_PreCirc_RCA18hr_Cy3Adapt_20x_pos02_fig03_Cy3_Exp0.2-EM40.tif|Original]]) || | |- |[[File:S1_washPBS3times_PreCirc_RCA18hr_Cy3Adapt_20x_pos02_fig04_BF_Exp0.2-EM00.jpg|300px]] || |[[File:S1_washPBS3times_PreCirc_RCA18hr_Cy3Adapt_20x_pos02_fig03_Cy3_Exp0.2-EM40.jpg|300px]] || |[[File:Composite_fig03-04.jpg|300px]] |}
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