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=====RCA===== *RCA reaction time **RCA is usually done for 20 hour reaction time, which would be enough. (I need to calculate if the amount of primers and dNTPs in the reaction mix are enough though) **Jay is doing exchange of fresh phi29 after 8 hour incubation, but he said this is not necessary. *Evaporation issue for long time incubation **There is no evaporation issue (20hr incubation), based on the observations after finishing RCA so far. *30C incubation condition **Currently I've used Jeff's microscope chamber for 30C incubation. **Try to use 30C oven, if I need to do. *phi29 concentration **Jay said rolonies will be plenty even if low concentration of phi29 is used, thus this is not a problem *After doing those two validation - RT, and Circularization, I'll compare between RCA and NTC(without phi29), and different reaction time such as 1hr, 5hr, 10hr, 20hr.
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