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=Test Mouse Embryo Pre-Processing Protocol for DARTFISH= *[[Matt:LabNotes/2015-6-23 | 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 *[[Matt:LabNotes/2015-7-6 | 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 ==ISH Protocol== *[[Media:ChiLab_Embryo_ISH_Protocol.PDF | 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 | 450px]] **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 #*Accidentally aspirated embryo again #*This form factor won't work for DARTFISH, too unreliable for exchanging solutions and will not work for 5 cycles of dye hybridization/stripping <!--#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 #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)-->
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