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=====Rolony test with using pre-circulated DNA===== *This test was done at [[Hosuk:LabNotes/2013-4-24|04/24]], but try one more time. *Since I've suspected that the binding between BS(PEG)9 and aminoallyl dUTP doesn't work well, so the rolonies could be washed out while washing step between after RCA and before adding BS(PEG)9, *3 dishes will be prepared to check this. *Detail procedure **Prepare pre-circularized DNA using 20 uL reaction in the tube ***Two identical 20uL reaction ***12pmole of custom ssDNA(50bp) in each tube **Add pre-circularized DNA in 3 dishes (S4, S5, S6) ***S4 : 3uL of Linear ssDNA + 47uL H2O (6 pmole of DNA) ***S5 : 10uL of pre-circularized DNA + 40uL H2O (6 pmole of DNA) ***S6 : 10uL of pre-circularized DNA + 40uL H2O (6 pmole of DNA) **Run RCA **After RCA ***S4 : Aspirate, then wash with PBS once, then add BS(PEG)9 ***S5 : Aspirate, then add BS(PEG)9 ***S6 : Aspirate, then wash with PBS once, then add BS(PEG)9 **block with 1M Tris pH 8.0 for 30min. **Aspirate, wash with PBS once **Prepare 100uM of adapter sequence Cy5-TCTCGGGAACGCTGAAGA ***2uL of 100uM probes in 200uL 2x SSC ***Pre-heated probe solution between 60C and 80C for 5min ***Add to cells at RT, and let it cool down over 5min ***Wash with 2x SSC (2mL, but 200uL work too) twice, and image
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