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

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>Mzcai
(Created page with "=Test Mouse Embryo Pre-Processing Protocol for DARTFISH= * First Try failed due to white particles after RT **Used Embryoe #3 and #4 **Try one mor...")
 
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===Procedure===
===Procedure===
#Make PBT: 1X PBS + 0.1% Triton X-100
#Make PBT: 1X PBS + 0.1% Triton X-100
#Rehydrate embryo #7 in 200ul PCR tube with attached caps
#Rehydrate embryo #7 in 2ml original tube
#*75% MeOH in PBT for 10min rotating
#*75% MeOH in PBT for 10min rotating
#*50% MeOH in PBT for 10min rotating
#*50% MeOH in PBT for 10min rotating
#**Embryo may have split in two? Maybe TX-100 concentration was too high in the 50% MeOH solution and needs to be remade
#*25% 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 tube
#*PBT for 10min rotating twice and add PBT to a second empty 2ml centrifuge tube
#Wash with 1X PBS three times for both tubes
#Wash with 1X PBS three times for both tubes
#Make RT Mix (2X volume) '''on ice'''
#Make RT Mix (2X volume) '''on ice'''
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#*M-MuLV RTase 100U/ul- 20ul
#*M-MuLV RTase 100U/ul- 20ul
#Add 200ul RT Mix to embryo tube and empty tube and incubate for 10min at 4C and then incubate overnight at 37C
#Add 200ul RT Mix to embryo tube and empty tube and incubate for 10min at 4C and then incubate overnight at 37C
 
#Wash with 1X PBS once
<!--#Wash with 1X PBS twice
#Add 200ul cold BS(PEG)9 (4ul stock BS(PEG)9 + 196ul 1X PBS prepared on ice) to sample and incubate 1hr at RT
#Wash with 1X PBS twice
#Add 200ul 1M Tris (pH 8.0) for 30min at RT
#Wash with 1X PBS twice
#Add RNase H mix and incubate 1hr at 37C
#Add RNase H mix and incubate 1hr at 37C
#*H2O - 168ul
#*H2O - 168ul
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#*Riboshredder - 2ul
#*Riboshredder - 2ul
#*RNase H - 10ul
#*RNase H - 10ul
-->
#Wash with nuclease-free H2O twice
#*ASPIRATED EMBRYO! :'(
<!--#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
#Add 200ul (1ul 200uM FISSEQ_RCA + 199ul 2X SSC + 30% formamide) and incubate 1hr at 60C
#Aspirate and wash with 2X SSC, then 1X SSC, and finally 1X PBS
#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
#Add RCA mix and incubate at 30C overnight (~14hrs)-->

Latest revision as of 23:59, 7 July 2015

Test Mouse Embryo Pre-Processing Protocol for DARTFISH[edit]

ISH Protocol[edit]

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

Fixed samples[edit]

FISSEQ[edit]

  • 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[edit]

  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
    • ASPIRATED EMBRYO! :'(