Hosuk:LabNotes/2013-6-10
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Training Day 1[edit]
- 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[edit]
- 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)