Hosuk:LabNotes/2014-5-7

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Revision as of 18:30, 7 May 2014 by >Hosuki78
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Test : +/- a-dUTP, and long RT incubation at 4C


Procedure

  • 05/06
    1. 10% Formalin for 15min at RT(25C) --> done at CERC
    2. Wash with PBS 3 times --> done at CERC
    3. Move samples to the lab at PFBH
    4. Add 0.25% Triton X-100 in DEPC-PBS and incubate for 10min at room temp.
    5. mean while prepare RT mix and put the mix on an ice
    6. Aspirate 0.25% Triton X-100 in DEPC-PBS and wash with PBS 3 times
    7. Add RT mix and put in 4C, incubate for 15min. (1hr. for other 2 sample of target specific primers)
    8. Move samples to 37C and incubate for 12hr.


  • 05/07
    1. Wash with PBS once
    2. Prepare 18uL of BS(PEG)9 in 782uL PBS (for 4 dishes, volume per each dish is 200uL)
    3. add 200uL BS(PEG)9 mix, incubate for 1hr at RT
    4. Wash with PBS once, add Tris pH 8.0, incubate for 30min
    5. RNase H mix : 2 ul Riboshredder (Epicentre)+ 10 ul RNase H(Enzymatics) in 20 ul RNase H buffer(Enzymatics) + 168 uL H2O
    6. Add RNase H mix, incubate for 1hr. at 37C