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

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Line 31: Line 31:
#*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
Line 39: Line 41:
#*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]

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 #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
  3. 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