Hosuk:LabNotes/2014-5-7: Difference between revisions

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*#add 200uL BS(PEG)9 mix, incubate for 1hr at RT
*#add 200uL BS(PEG)9 mix, incubate for 1hr at RT
*#Wash with PBS once, add Tris pH 8.0, incubate for 30min
*#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
*#Add RNase H mix, incubate for 1hr. at 37C
*#
*#*RNase H mix : 2 ul Riboshredder (Epicentre)+ 10 ul RNase H(Enzymatics) in 20 ul RNase H buffer(Enzymatics) + 168 uL H2O
*#Wash with nucleaus-free H2O twice
*#Add CircLigase II mix, incubate at 60C for 3hr.
*#*H2O 128uL
*#*Buffer 20uL
*#*50mM MnCl2 10uL
*#*5M Betaine 40uL
*#*CircLigaseII 2uL
*#RCA primer, final concentration : 0.5uM in 200uL (2x SSC + 30% Formamide) at 60C for 1hr.

Revision as of 20:15, 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)
    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. Add RNase H mix, incubate for 1hr. at 37C
      • RNase H mix : 2 ul Riboshredder (Epicentre)+ 10 ul RNase H(Enzymatics) in 20 ul RNase H buffer(Enzymatics) + 168 uL H2O
    6. Wash with nucleaus-free H2O twice
    7. Add CircLigase II mix, incubate at 60C for 3hr.
      • H2O 128uL
      • Buffer 20uL
      • 50mM MnCl2 10uL
      • 5M Betaine 40uL
      • CircLigaseII 2uL
    8. RCA primer, final concentration : 0.5uM in 200uL (2x SSC + 30% Formamide) at 60C for 1hr.