Matt:LabNotes/2016-7-14: Difference between revisions
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>Mzcai (Created page with "=FISSEQ on BA8 sections with PA gel + Acrydite/BS(PEG)9= *Try FISSEQ to see a "baseline" of number of rolonies *Gel mix and protocol is same as [[Matt:LabNotes/2016-6-28#Gel_M...") |
>Mzcai mNo edit summary |
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Line 84: | Line 84: | ||
| Total||200 | | Total||200 | ||
|} | |} | ||
#Incubate 10min at 4C and then ~18hr at 37C | #Incubate 10min at 4C and then ~18hr at 37C | ||
===Day 2=== | ===Day 2=== | ||
#Wash with 1X PBS once | #Wash with 1X PBS once | ||
#Add 200ul cold BS(PEG)9 (4ul stock BS(PEG)9 + 196ul 1X PBS) and incubate 1hr at RT | #Add 200ul cold BS(PEG)9 (4ul stock BS(PEG)9 + 196ul 1X PBS) and incubate 1hr at RT | ||
#Wash with 1X PBS twice | #Wash with 1X PBS twice | ||
*[[File: | *[[File:|250px]] | ||
#Add 1M Tris pH 8.0 and incubate 30min at RT | #Add 1M Tris pH 8.0 and incubate 30min at RT | ||
#Wash with 1X PBS twice | #Wash with 1X PBS twice | ||
Line 96: | Line 95: | ||
#*Add 200ul RNase Mix (20ul RNase H 10X Buffer + 2ul Riboshredder + 10ul RNase H + 168ul H2O) and incubate 1hr at 37C | #*Add 200ul RNase Mix (20ul RNase H 10X Buffer + 2ul Riboshredder + 10ul RNase H + 168ul H2O) and incubate 1hr at 37C | ||
#Wash with nf-H2O twice | #Wash with nf-H2O twice | ||
#Prepare | #Prepare Circligase mix on ice | ||
{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''Component''' | | align="center" style="background:#f0f0f0;"|'''Component''' | ||
| align="center" style="background:#f0f0f0;"|'''Volume''' | | align="center" style="background:#f0f0f0;"|'''Volume''' | ||
|- | |- | ||
| DEPC-H2O|| | | DEPC-H2O||128 | ||
|- | |- | ||
| | | CircLigase II Buffer 10X||20 | ||
|- | |- | ||
| | | MnCl2 50mM||10 | ||
|- | |- | ||
| | | Betaine 5M||40 | ||
|- | |- | ||
| Total|| | | CircLigase II 100U/ul||2 | ||
|- | |||
| Total||200 | |||
|} | |} | ||
#Add mix to sample and incubate for | #Add mix to sample and incubate for 3hr at 60C | ||
#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 | |||
#Wash with 1X PBS | #Prepare RCA reaction mix on ice | ||
#Add | |||
# | |||
# | |||
{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''Component''' | | align="center" style="background:#f0f0f0;"|'''Component''' | ||
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| Total||200 | | Total||200 | ||
|} | |} | ||
===Day | #Add RCA mix and incubate at 30C overnight (~15hrs)<!-- | ||
===Day 3=== | |||
#Wash with 1X PBS once | #Wash with 1X PBS once | ||
#Add 200ul BS(PEG)9 (4ul BS(PEG)9 + 196ul 1X PBS) and incubate at RT for 1hr | #Add 200ul BS(PEG)9 (4ul BS(PEG)9 + 196ul 1X PBS) and incubate at RT for 1hr |
Revision as of 18:12, 15 July 2016
FISSEQ on BA8 sections with PA gel + Acrydite/BS(PEG)9
- Try FISSEQ to see a "baseline" of number of rolonies
- Gel mix and protocol is same as this with BS(PEG)9
Component | Volume |
40% AB 199:1 Mix | 6.25 |
10% BSA | 1 |
1mM Acryd/Amine Linker | 6.25 |
H2O | 33.5 |
5% TEMED | 1 |
5% APS | 1 |
BS(PEG)9 | 1 |
Total | 50 |
Protocol
Day 1
- Prepare a plastic culture dish with 12x17mm hole, tweezers, large plastic dish, and 50C hot plate
- EtOH and UV sterilize
- Use 4% PFA in 1X PBS from 7/6/2016
- Take out BA8 section from -80C and dry on 50C hot plate for 3min
- Submerge coverslip in 5ml 4% PFA in 6 well culture plate for 15min at 37C
- Wash twice with cold 1X SSPE by submerging
- Add 0.25% TX-100 in 2XSSPE and incubate 10min at RT
- Wash with cold nf-H2O three times and check for degradation
- Add 0.01% Pepsin in 0.1N HCl and incubate for 90sec at 37C
- 2ul 1%Pepsin + 10ul 2N HCl + 188ul H2O
- Wash with nf-1X PBS three times and check for degradation
- Aspirate any liquid and place a glass slide with 50um spacer over the coverslip with tissue
- Secure in plastic jig
- Add 50ul gel casting mix filtered and degassed
- 5% gel with 250uM Acrydite-Amine linker and 5mM BS(PEG)9
- Seal in plastic bag and vacuum out air before filling with argon
- Let sit at RT for 30min
- Aspirate non-polymerized gel and wash once with 1X PBS
- Attach coverslip to bottom of petri dish
- Aspirate non-polymerized gel and wash once with 1M Tris and once with cold 1X SSPE
- Prepare Reverse Transcription Mix on ice and add
Components | Volume |
H2O | 159 |
10X M-MuLV Buffer | 20 |
25mM dNTP | 2 |
4mM aa-dUTP | 2 |
100uM FISSEQ_RT primer | 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 (4ul stock BS(PEG)9 + 196ul 1X PBS) and incubate 1hr at RT
- Wash with 1X PBS twice
- [[File:|250px]]
- Add 1M Tris pH 8.0 and incubate 30min at RT
- Wash with 1X PBS twice
- RNA Removal
- Add 200ul RNase Mix (20ul RNase H 10X Buffer + 2ul Riboshredder + 10ul RNase H + 168ul H2O) and incubate 1hr at 37C
- Wash with nf-H2O twice
- Prepare Circligase mix on ice
Component | Volume |
DEPC-H2O | 128 |
CircLigase II Buffer 10X | 20 |
MnCl2 50mM | 10 |
Betaine 5M | 40 |
CircLigase II 100U/ul | 2 |
Total | 200 |
- Add mix to sample and incubate for 3hr at 60C
- 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
Component | Volume |
H2O | 174 |
10X Phi29 Buffer | 20 |
25mM dNTP | 2 |
4mM aa-dUTP | 2 |
Phi 29 (low conc) | 2 |
Total | 200 |
- Add RCA mix and incubate at 30C overnight (~15hrs)