Matt:LabNotes/2015-7-7: Difference between revisions
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#*PBT for 10min rotating twice and add PBT to a second empty 2ml centrifuge 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 '''on ice''' | #*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 | #*H2O - 157ul | ||
#*M-MuLV RT Buffer 10X - 20ul | #*M-MuLV RT Buffer 10X - 20ul | ||
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#*RNase Inhibitor 40U/ul- 2ul | #*RNase Inhibitor 40U/ul- 2ul | ||
#*M-MuLV RTase 100U/ul- 10ul | #*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 | #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 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 | #Add 200ul cold BS(PEG)9 (4ul stock BS(PEG)9 + 196ul 1X PBS prepared on ice) to sample and incubate 1hr at RT |
Latest revision as of 01:12, 8 July 2015
Test Mouse Embryo Pre-Processing Protocol for DARTFISH[edit]
- 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[edit]
- Protocol.pdf
- Fixed, dehydrated in methanol, and stored in -20C
- Rehydrated and permeabilized with ProK for ISH
Fixed samples[edit]
- File:MERT 2015-05-14 E7.5 FISSEQ.JPG
- Got 7 embryos each in a 2ml tube and stored in -20C
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]
- 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