Matt:LabNotes/2016-7-19: Difference between revisions
Jump to navigation
Jump to search
>Mzcai m (→FISSEQ) |
>Mzcai mNo edit summary |
||
(3 intermediate revisions by the same user not shown) | |||
Line 76: | Line 76: | ||
|} | |} | ||
#Add 100ul to each and incubate 10min at 4C and then ~16hr at 37C | #Add 100ul to each and incubate 10min at 4C and then ~16hr at 37C | ||
===Day 2=== | ===Day 2=== | ||
#Wash with 1X PBS once | #Wash with 1X PBS once | ||
#Add | #Add 100ul 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 | #Wash with 1X PBS twice | ||
#Add 200ul 1M Tris (pH 8.0) for 30min at RT | #Add 200ul 1M Tris (pH 8.0) for 30min at RT | ||
Line 96: | Line 96: | ||
#*CircLigase II 100U/ul - 2ul | #*CircLigase II 100U/ul - 2ul | ||
#Wash with 1X PBS twice | #Wash with 1X PBS twice | ||
#*Lost many embryos due to sticking to pipette tip and unable to get off without scraping | |||
#*Only one CLARITY and one PACT embryo left | |||
#Add 200ul (1ul 200uM FISSEQ_RCA + 199ul 2X SSC + 30% formamide) and incubate 1hr at 60C | #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 | #Aspirate and wash with 2X SSC, then 1X SSC, and finally 1X PBS | ||
#*PACT embryo stuck to pipette tip so cut off the tip and put in tube for incubation | |||
#Prepare RCA reaction mix '''on ice''' | #Prepare RCA reaction mix '''on ice''' | ||
#*H2O - 174ul | #*H2O - 174ul | ||
Line 104: | Line 107: | ||
#*aa-dUTP 4mM - 2ul | #*aa-dUTP 4mM - 2ul | ||
#*Phi29 DNA polymerase 100U/ul - 2ul | #*Phi29 DNA polymerase 100U/ul - 2ul | ||
#Add RCA mix and incubate at 30C overnight (~ | #Add RCA mix and incubate at 30C overnight (~12hrs) | ||
===Day 3=== | ===Day 3=== | ||
#Wash with 1X PBS once | #Wash with 1X PBS once | ||
#Add 200ul cold BS(PEG)9 ( | #*Could not find embryo stuck to pipette tip | ||
#*Only one CLARITY embryo left | |||
#Add 200ul cold BS(PEG)9 (4ul stock BS(PEG)9 + 196ul 1X PBS prepared on ice) to sample and incubate 1hr at RT | |||
#Wash with 1X PBS twice | #Wash with 1X PBS twice | ||
#Add 200ul 1M Tris (pH 8.0) for 30min at RT | #Add 200ul 1M Tris (pH 8.0) for 30min at RT | ||
#Wash with 1X PBS twice | #Wash with 1X PBS twice | ||
====Imaging==== | ====Imaging==== | ||
Line 123: | Line 122: | ||
#Incubate for 10min at RT | #Incubate for 10min at RT | ||
#Wash with 2X SSC twice | #Wash with 2X SSC twice | ||
# | #Mount between coverslip and slide with standard mounting media and seal with nail polish | ||
*Essentially zero Cy3 signal in embryo |
Latest revision as of 00:55, 26 July 2016
CLARITY and PACT for FISSEQ in Mouse Embryo E7.5[edit]
- Need more permeabilization to get enzymes and reagents deeper into tissue
- To permeabilize will remove lipids with detergent in long incubation
- Need to embed tissue in hydrogel matrix to create structure that will survive detergent
Experiment[edit]
PACT Protocol[edit]
- Fix embryo in 4% PFA at 37C for 15min
- Make 4mL PACT solution on ice
- 4% acrylamide in 1X PBS plus 0.25% VA-044 (degassed)
- 3.584mL MilliQ H2O + 0.448mL 10X PBS + 0.448mL 40% acrylamide (cold) + 0.0112g 0.25% VA-044 (cold)
- Wash with 1X PBS
- Incubate embryo in solution at 4C overnight (20hrs)
- Polymerize gel by incubating at 37C for 3hr
- Did not degas under Argon gas but can do next time if not polymerized enough
- Make 500mL 8% SDS solution
- Could use 0.2 M boric acid or 0.01 M PBS (1X)
- 6.183g boric acid, 200mL MilliQ H2O, pH with NaOH to pH=8.5, MilliQ H2O to 300mL total, then add 200mL 20% SDS
- 50mL = 5mL 10X PBS + 20mL 20% SDS + 25mL H2O
- Remove embryo from gel
- Pipette away surrounding hydrogel and wash with 1X PBS
- Incubate in 8% SDS at 37C on rotator for overnight
- Tissue will not be fully clear until put into refractive index matching media
- Wash embryo in PBS + 0.1% TX-100 at 37C on a rotator for 6 hrs
- Need to wash away SDS or it will precipitate forming cloudy spots
CLARITY Protocol[edit]
- Make 4mL CLARITY solution on ice
- 4% PFA, 0.95% acrylamide, 0.05% bis-acrylamide, 0.25% VA-044, 1X PBS (degassed)
- 0.5mL 40% PFA + 0.125mL 40% 19:1 acrylamide:bis + 0.0125g VA-044 + 0.5mL 10X PBS + 3.875mL H2O
- Incubate embryo in solution at 4C overnight (20hrs)
- Polymerize gel by incubating at 37C for 3hr
- Make 500mL 8% SDS solution
- Could use 0.2 M boric acid or 0.01 M PBS (1X)
- 6.183g boric acid, 200mL MilliQ H2O, pH with NaOH to pH=8.5, MilliQ H2O to 300mL total, then add 200mL 20% SDS
- 50mL = 5mL 10X PBS + 20mL 20% SDS + 25mL H2O
- Remove embryo from gel
- Pipette away surrounding hydrogel and wash with 1X PBS
- Roll on kimwipe to remove bits of gel
- Incubate in 8% SDS at 37C on rotator for overnight
- Tissue will not be fully clear until put into refractive index matching media
- Wash embryo in PBS + 0.1% TX-100 at 37C on a rotator for 6 hrs
- Need to wash away SDS or it will precipitate forming cloudy spots
FISSEQ[edit]
- 4 tdTomato positive embryos were taken by Paola for confocal/two-photon microscopy test
- For tdTomato negative embryos do FISSEQ
- 4 CLARITY embryos and 2 PACT
Day 1[edit]
- Wash with PBS three times
- Prepare 3X 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 |
- Add 100ul to each and incubate 10min at 4C and then ~16hr at 37C
Day 2[edit]
- Wash with 1X PBS once
- Add 100ul 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
- Lost many embryos due to sticking to pipette tip and unable to get off without scraping
- Only one CLARITY and one PACT embryo left
- 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
- PACT embryo stuck to pipette tip so cut off the tip and put in tube for incubation
- 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 (~12hrs)
Day 3[edit]
- Wash with 1X PBS once
- Could not find embryo stuck to pipette tip
- Only one CLARITY embryo left
- Add 200ul cold BS(PEG)9 (4ul stock BS(PEG)9 + 196ul 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
Imaging[edit]
- Add 200ul of 0.5uM FISSEQ_Adpt Cy3 in 2X SSC + 30% formamide pre-heated to 75C
- Incubate for 10min at RT
- Wash with 2X SSC twice
- Mount between coverslip and slide with standard mounting media and seal with nail polish
- Essentially zero Cy3 signal in embryo