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=====Circularization===== *Most suspicious step *Reaction time **In the manual, 1 hour will be enough, so 2 hour reaction time would not be a problem. *Mineral oil cover to prevent evaporation **I've tried both mineral oil and wet towel for 2 hour, 60C incubation **Result (observation of rolonies after RCA) was same, thus mineral oil cover, washing step are not a problem. *The amount of CircLigase enzyme **The volume of CircLigase II enzyme was 2uL in the very first protocol that Jay sent. **However, in the phone call at 02-07, Jay said 2uL is ok because of expensive cost of the enzyme. **Original protocol in the manual of CircLigase II is 100 U per 20uL reaction volume, which means '''5uL''' for 100uL reaction that currently I've used. **'''I will try 5uL of CircLigase II enzyme''' and do RCA and observe rolonies if there is any difference. *Circularization test **I'll use '''50 bp sequence having the adapter sequence''' that I'm using (TCTCGGGAACGCTGAAGA) and circularization, RCA and observe how rolonies are seen. **(1) order 50 bp oligo ***5' CAA GCA GAA GAC GGC A[TC TCG GGA ACG CTG AAG A]CT CGG CAT TCC TGC TG 3' (--> 50bp, sequence in the [] is the adapter sequence ) **(2) Circularization test in the test tube using CircLigase II enzyme, gel running for verification **(3) Inject circular templates that are made from tube in the fixed cells, the RCA '''--> need to ask detail method how to hybridize circular template inside cells''' because there is not aminoallyl dUTP. **(4) Hybridize linear 50bp oligo in the fixed cells, circularization and RCA '''--> need to ask detail method how to hybridize 50 bp oligo inside cells''' because there is not aminoallyl dUTP. **(5) Compare rolonies (3), (4), and NTC (non CircLigase sample)
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