Hosuk:LabNotes/2014-5-7
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Test : +/- a-dUTP, and long RT incubation at 4C
1st Try
- 8 MatTek dishes - PGP1F (05/06)
- 10% Formalin for 15min at RT(25C),
- 0.25% Triton X-100 for 10min at RT
- 0.1N HCL incubation at RT
- BUT! Samples were gone, destroyed!
- upto 0.1N HCL process seemed too harsh
- Let’s just do Triton X-100 and run reverse transcript!
2nd Try
- With only 4 sample (05/07)
- Sample 1 : +a-dUTP, Random hexamer, 15min incubation at 4C
- Sample 2 : -a-dUTP, Random hexamer, 15min incubation at 4C
- Sample 3 : +a-dUTP, ACTB, RAB7A primer, 1hr. incubation at 4C
- Sample 4 : -a-dUTP, ACTB, RAB7A primer, 1hr. incubation at 4C
Procedure
- Follow the latest protocol (Nature protocol manuscript)
- 10% Formalin for 15min at RT(25C) --> done at CERC
- Wash with PBS 3 times --> done at CERC
- Move samples to the lab at PFBH
- Add 0.25% Triton X-100 in DEPC-PBS and incubate for 10min at room temp.
- mean while prepare RT mix and put the mix on an ice
- Aspirate 0.25% Triton X-100 in DEPC-PBS and wash with PBS 3 times
- Add RT mix and put in 4C, incubate for 15min. (1hr. for other 2 sample of target specific primers)