Hosuk:LabNotes/2014-5-7
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Test : +/- a-dUTP, and long RT incubation at 4C
- Continued from 05/06
Procedure
- 05/06
- 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)
- Move samples to 37C and incubate for 12hr.
- 05/07
- Wash with PBS once
- Prepare 18uL of BS(PEG)9 in 782uL PBS (for 4 dishes, volume per each dish is 200uL)
- add 200uL BS(PEG)9 mix, incubate for 1hr at RT
- Wash with PBS once, add Tris pH 8.0, incubate for 30min
- RNase H mix : 2 ul Riboshredder (Epicentre)+ 10 ul RNase H(Enzymatics) in 20 ul RNase H buffer(Enzymatics) + 168 uL H2O
- Add RNase H mix, incubate for 1hr. at 37C