Matt:LabNotes/2015-7-6: Difference between revisions
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>Mzcai m (→Procedure) |
>Mzcai m (→Procedure) |
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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
- First Try failed due to white particles after RT
- Used Embryoe #3 and #4
- Try one more time with a control (no embryo) to see if white particles are degraded embryo or precipitate
ISH Protocol
- Protocol.pdf
- Fixed, dehydrated in methanol, and stored in -20C
- Rehydrated and permeabilized with ProK for ISH
Fixed samples
- File:MERT 2015-05-14 E7.5 FISSEQ.JPG
- Got 7 embryos each in a 2ml tube and stored in -20C
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
- Make PBT: 1X PBS + 0.1% Triton X-100
- 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
- Wash with 1X PBS three times for both tubes
- 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
- 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
- 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
- H2O - 168ul
- RNase H Buffer - 20ul
- Riboshredder - 2ul
- RNase H - 10ul
- Wash with nuclease-free H2O twice
- 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)