Hosuk:LabNotes/2014-5-7: 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 (05/06) **10% Formalin for 15min at RT(25C), **0.2...")
 
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===Test : +/- a-dUTP, and long RT incubation at 4C===
===Test : +/- a-dUTP, and long RT incubation at 4C===
====1st Try====
*Continued from [[Hosuk:LabNotes/2014-5-6|05/06]]
*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====
====Procedure====
*Follow the latest protocol (Nature protocol manuscript)
*05/06
*#10% Formalin for 15min at RT(25C) --> done at CERC
*#10% Formalin for 15min at RT(25C) --> done at CERC
*#Wash with PBS 3 times --> done at CERC
*#Wash with PBS 3 times --> done at CERC
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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.
*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
*

Revision as of 17:53, 7 May 2014

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)
  • add 200uL BS(PEG)9 mix, incubate for 1hr at RT