Matt:LabNotes/2016-3-8: Difference between revisions
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>Mzcai (Created page with "=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 f...") |
>Mzcai m (→Day 1) |
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#Wash with cold nf-H2O twice | #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 | #Add 200ul 0.01% pepsin in 0.01N HCl (2ul 1%Pepsin + 10ul 2N HCl + 188ul H2O) for 1min at 37C | ||
#Wash with 2ml | #Wash with 2ml nf-PBS three times | ||
#Prepare 2X Reverse Transcription Mix '''on ice''' | #Prepare 2X Reverse Transcription Mix '''on ice''' | ||
{| {{table}} | {| {{table}} |
Revision as of 01:34, 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