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*[[Hosuk:Lab_Notes|LabNote]] ===Rolony, 13th=== *Check point *#2hr vs. 4hr CircLigase II reaction *#Strip and re-hybridize Cy5 probe *Start at 06/29 **Use cell dishes fixed at 07/07 --> Cells were not plenty… need to thaw new one **GC, lysine coated dish **Run RT : 5:00pm 07/07 ~ 9:00am 07/08 --> ~15hr. **Run CircLigase II : S1 --> 1:30pm 07/08 ~ 3:30pm 07/08 --> 2hr. **Run CircLigase II : S2 --> 11:30am 07/08 ~ 3:30pm 07/08 --> 4hr. **Run RCA : 4:30 pm 07/08 ~ 12:30pm 07/09 --> ~20hr. ====Result==== *Not as many as before **It could be something wrong while fixing (10% formalin once) or cells were not good. **For the next time, I’ll do twice fixation. (37% and then 10% formalin), and cells need to be full **Rolonies were found inside nucleus more than other area. --> it might cause from not many cells or one time fixation. *2hr, 4hr were not much different! *After striping Cy3 probe, there was no signal from Cy3 channel. *And after re-hybridizing Cy5 probe, rolonies were nicely seen through Cy5 channel. *Rolonies were pretty stable. *Strip protocol: *#80% formamide in 2x SSC heated in 60C oven for 5min *#Add 200uL of 80% formamide on cells, incubate at RT for 10min. *#Wash with H2O twice, and store in PBS ====2hr vs. 4hr CircLigase II==== [[File:S1-2hr_S2-4hrCirc_20x-NA0.8_3uLPhi29_exp0.1_EM40.png|600px]] ====After strip==== [[File:S2-4hrCirc_AfterStrip_20x-NA0.8_3uLPhi29_exp0.1_EM40.png|900px]] ====After re-hybridizing Cy5 probe==== [[File:S2-4hrCirc_AfterStripAndReHybriCy5_20x-NA0.8_3uLPhi29_exp0.1_EM40.png|900px]]
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