Hosuk:LabNotes/2014-5-6: Difference between revisions

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(Created page with "*LabNote ===Test : +/- a-dUTP, and long RT incubation at 4C=== ====1st Try==== *8 MatTek dishes - PGP1F (05/05) **10% Formalin for 15min at RT(25C), **0.2...")
 
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*#Aspirate 0.25% Triton X-100 in DEPC-PBS and wash with PBS 3 times
*#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)
*#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.

Latest revision as of 17:51, 7 May 2014

Test : +/- a-dUTP, and long RT incubation at 4C[edit]

1st Try[edit]

  • 8 MatTek dishes - PGP1F (05/05)
    • 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[edit]

  • With only 4 sample (05/06)
    • 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[edit]

  • Follow the latest protocol (Nature protocol manuscript)
    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.