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

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>Mzcai
(Created page with "=Test Mouse Embryo Pre-Processing Protocol for DARTFISH= * First Try failed due to white particles after RT **Used Embryo #3 and #4 **White partic...")
 
>Mzcai
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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 (2X volume) '''on ice'''
#Make RT Mix '''on ice'''
#*H2O - 314ul
#*H2O - 157ul
#*M-MuLV RT Buffer 10X - 40ul
#*M-MuLV RT Buffer 10X - 20ul
#*dNTP 25mM- 4ul
#*dNTP 25mM- 2ul
#*aa-dUTP 2mM- 8ul
#*aa-dUTP 2mM- 4ul
#*FISSEQ_RT primer (nonamer) 100uM - 10ul
#*FISSEQ_RT primer (nonamer) 100uM - 5ul
#*RNase Inhibitor 40U/ul- 4ul
#*RNase Inhibitor 40U/ul- 2ul
#*M-MuLV RTase 100U/ul- 20ul
#*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

Revision as of 00:22, 8 July 2015

Test Mouse Embryo Pre-Processing Protocol for DARTFISH

ISH Protocol

  • Protocol.pdf
    • Fixed, dehydrated in methanol, and stored in -20C
    • Rehydrated and permeabilized with ProK for ISH

Fixed samples

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

  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
  4. 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
  5. Add 200ul RT Mix to embryo tube and empty tube and incubate for 10min at 4C and then incubate overnight at 37C