Matt:LabNotes/2015-7-6: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Mzcai
>Mzcai
Line 47: Line 47:
#Wash with nuclease-free H2O twice
#Wash with nuclease-free H2O twice
#Add CircLigase mix and incubate 1hr at 60C
#Add CircLigase mix and incubate 1hr at 60C
#*
#*H2O - 128ul
#*
#*CircLigase Buffer 10X - 20ul
#*
#*MnCl2 50mM - 10ul
#*
#*Betaine 5M - 40ul
#*
#*CircLigase II 100U/ul - 2ul
#Wash with 1X PBS twice
#Wash with 1X PBS twice
#Add 200ul (1ul 200uM FISSEQ_RCA + 199ul 2X SSC + 30% formamide) and incubate 1hr at 60C
#Add 200ul (1ul 200uM FISSEQ_RCA + 199ul 2X SSC + 30% formamide) and incubate 1hr at 60C

Revision as of 22:11, 7 July 2015

Test Mouse Embryo Pre-Processing Protocol for DARTFISH

ISH Protocol

  • Protocol.pdf
    • Fixed, dehydrated in methanol, and stored in -20C
    • Rehydrated and permeabilized with ProK for ISH

Fixed samples

FISSEQ

  • To save padlock probes will test fixation/permeabilization sample conditions with FISSEQ
  • Will use universal probe for all rolonies (FISSEQ_Adpt Cy3?) to check
  • To image, sandwich embryo between glass slide and coverslip

Procedure

  1. Make PBT: 1X PBS + 0.1% Triton X-100
  2. Rehydrate embryo #7 in 2ml original tube
    • 75% MeOH in PBT for 10min rotating
    • 50% MeOH in PBT for 10min rotating
    • 25% MeOH in PBT for 10min rotating
    • PBT for 10min rotating twice and add PBT to a second empty 2ml centrifuge tube
  3. Wash with 1X PBS three times for both tubes
  4. Make RT Mix (2X volume) on ice
    • H2O - 314ul
    • M-MuLV RT Buffer 10X - 40ul
    • dNTP 25mM- 4ul
    • aa-dUTP 2mM- 8ul
    • FISSEQ_RT primer (nonamer) 100uM - 10ul
    • RNase Inhibitor 40U/ul- 4ul
    • M-MuLV RTase 100U/ul- 20ul
  5. Add 200ul RT Mix to embryo tube and empty tube and incubate for 10min at 4C and then incubate overnight at 37C
  6. Wash with 1X PBS once
  7. Add 200ul cold BS(PEG)9 (4ul stock BS(PEG)9 + 196ul 1X PBS prepared on ice) to sample and incubate 1hr at RT
  8. Wash with 1X PBS twice
  9. Add 200ul 1M Tris (pH 8.0) for 30min at RT
  10. Wash with 1X PBS twice
  11. Add RNase H mix and incubate 1hr at 37C
    • H2O - 168ul
    • RNase H Buffer - 20ul
    • Riboshredder - 2ul
    • RNase H - 10ul
  12. Wash with nuclease-free H2O twice
  13. Add CircLigase mix and incubate 1hr at 60C
    • H2O - 128ul
    • CircLigase Buffer 10X - 20ul
    • MnCl2 50mM - 10ul
    • Betaine 5M - 40ul
    • CircLigase II 100U/ul - 2ul
  14. Wash with 1X PBS twice
  15. Add 200ul (1ul 200uM FISSEQ_RCA + 199ul 2X SSC + 30% formamide) and incubate 1hr at 60C
  16. Aspirate and wash with 2X SSC, then 1X SSC, and finally 1X PBS
  17. Prepare RCA reaction mix on ice
    • H2O - 174ul
    • Phi29 Buffer 10X - 20ul
    • dNTP 25mM - 2ul
    • aa-dUTP 4mM - 2ul
    • Phi29 DNA polymerase 100U/ul - 2ul
  18. Add RCA mix and incubate at 30C overnight (~14hrs)