Hosuk:LabNotes/2013-6-18
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Rolony, 9th
- Try the method that I’ve learned from Jonathan and Jay
- 2uL Riboshredder + 10uL Rnase H + 20uL Rnase H buffer + 168uL H2O (Jonathan’s protocol)
- Use double volume of CircLigase reagent except CircLigase II (Jonathan’s protocol)
- Use Enzymatics’ Phi29 --> 1uL Phi29 (High concentration of Enzymatics) for RCA mix
- Use plastic bag with wet tissue for long time incubation
- Start at 06/16
- Use cell dishes fixed at 06/04
- S3 --> -RT, S4 --> +RT
- Run RT : 9:00pm 06/16 ~ 9:00am 06/17 --> 12hr.
- Run CircLigase II : 12:05am 06/17 ~ 4:05pm 06/17 --> 4hr.
- Run RCA : 5:00 pm 06/17 ~ 3: 10pm 06/18 --> 22hr.
Result
- S3, -RT sample didn't show any rolony signal.
- S4, +RT sample had not as many rolonies as what I expected!
- Low background, and no signal through FITC nor Cy5 channel though,
- Use sigma's Betaine because of running out of original betaine in CircLigase II kit, would it be reason? not sure!
- Need to try one more! using original protocol of CircLigase II!
epi FL
- -RT, 20x, NA=0.8
File:S3 -RT 20x-NA0.8 pos02.png
- +RT, 20x, NA=0.8
File:S4 +RT 20x-NA0.8 pos02.png
- +RT, 40x Oil, NA=1.3