Matt:LabNotes/2015-7-7
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Test Mouse Embryo Pre-Processing Protocol for DARTFISH
- First Try failed due to white particles after RT
- Used Embryo #3 and #4
- White particles probably pieces of embryo or else precipitates in buffer
- To avoid, don't spin down embryo
- To avoid, spin down buffer precipitates before pipetting buffer into mix
- Second Try failed due to aspirating embryo
- Used Embryo #7
- To avoid, try making sure embryo is in bottom corner of 2ml tube before aspirating solution
- Used Embryo #7
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 #6 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 on ice
- H2O - 157ul
- M-MuLV RT Buffer 10X - 20ul
- dNTP 25mM- 2ul
- aa-dUTP 2mM- 4ul
- FISSEQ_RT primer (nonamer) 100uM - 5ul
- RNase Inhibitor 40U/ul- 2ul
- M-MuLV RTase 100U/ul- 10ul
- Add 200ul RT Mix to embryo tube and empty tube and incubate for 10min at 4C and then incubate overnight at 37C