Matt:LabNotes/2016-3-8: Difference between revisions
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>Mzcai m (→Day 1) |
>Mzcai m (→Day 1) |
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#Incubate 10min at 4C and then ~18hr at 37C | #Incubate 10min at 4C and then ~18hr at 37C | ||
===Day 2=== | |||
#Wash with 1X PBS once | |||
#Add 200ul cold BS(PEG)9 (12ul stock BS(PEG)9 + 588ul 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 3hr 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) |
Revision as of 01:55, 9 March 2016
FISSEQ whole mount Mouse Embryo
Procedure
- Embryo is fresh and sitting in 1X PBS in 200ul PCR tube
Day 1
- Sterilize bench and tweezers with EtOH, RNaseZap
- Make fresh 4% PFA in PBS
- 10ml 16% PFA + 4ml 10X PBS + 26ml nfH2O
- Add Argon gas and seal with parafilm to keep it fresh longer
- Aspirate PBS from tube and add 200ul 4% PFA
- Incubate at 37C for 15min
- Wash twice using cold nf-SSPE
- Add 0.25% TX-100 in nf-2XSSPE for 5min at RT
- Wash with cold nf-H2O twice
- Add 200ul 0.01% pepsin in 0.01N HCl (2ul 1%Pepsin + 10ul 2N HCl + 188ul H2O) for 1min at 37C
- Wash with 2ml nf-PBS three times
- Prepare 2X Reverse Transcription Mix on ice
Components | Volume |
H2O | 157 |
10X M-MuLV Buffer | 20 |
25mM dNTP | 2 |
4mM aa-dUTP | 2 |
100uM FISSEQ_RT | 5 |
RNase Inhibitor | 2 |
M-MuLV RTase | 10 |
Total | 200 |
- Incubate 10min at 4C and then ~18hr at 37C
Day 2
- Wash with 1X PBS once
- Add 200ul cold BS(PEG)9 (12ul stock BS(PEG)9 + 588ul 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 3hr 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)