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*[[Hosuk:Lab_Notes|LabNote]] ====Training Day 1==== *Some of important things *Phi29 enzyme from '''Enzymatics''' is used. *Actually Jay likes to use (or trusts) Enzymatics, so almost all reagents are from Enzymatics. *The concentration of Phi29 of Enzymatics that Jonathan has used is '''100U/uL''' which is '''High concentration one''' from Enzymatics, and this concentration is the same as low concentration one from Epicentre. **'''1uL of phi29 in 200uL total mix''' **I could try 3uL of phi29 *RNase H step after RT is critical step, so do this carefully. *RNase H + Riboshredder mix is **'''10uL of RNase H''' + **'''2uL of Riboshredder''' + **'''20uL of 10x Buffer''' + **168uL of H2O *Try HeLa cells unless I get rolonies with PGP1F. *The sample (that I've got some signal at 05/29) doesn't seem to have actual rolonies, *There are no dots, just too much even signal distribution. *Jonathan fixed cells just using 10% Formamide **Aspirate media **Add 50uL of 10% Formamide (200uL for glass bottom) **Incubate for 15min **Wash with PBS three times **Store in Ethanol =====Rolony Construction===== *S1 : +RT, +CircLigase, +RCA *S2 : -RT (no RT enzyme), +CircLigase, +RCA *Jonathan's samples in 96 well (2 positive, 2 negative controls) *Jonathan fixed his cells in 96 well plate, using 10% Formalin and incubating 15min, without 37% Formalin. *HCL incubation for 1min. *Run RT for overnight (5:00pm)
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