Revision as of 02:43, 22 July 2016 by >Mzcai
DARTFISH on BA8 sections with PA gel + Acrydite/BS(PEG)9
Component
|
Volume
|
40% AB 199:1 Mix
|
6.25
|
10% BSA
|
1
|
Acryd/Amine Linker
|
25
|
H2O
|
14.75
|
5% TEMED
|
1
|
5% APS
|
1
|
BS(PEG)9
|
1
|
Total
|
50
|
- Half the volume because limited Acrydite/Amine linker left
Protocol
Day 1
- Prepare two plastic culture dishes with 12x17mm hole, tweezers, large plastic dish, and 50C hot plate
- Make fresh 4% PFA in 1X PBS
- 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
- Add 50ul gel casting mix filtered and degassed
- 5% gel with 500uM 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 N9 |
5
|
RNase Inhibitor |
2
|
M-MuLV RTase |
10
|
Total |
200
|
- Incubate 10min at 4C and then ~18hr at 37C
Day 2
- Gel does not look significantly swollen
- 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
- 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 Ampligase mix on ice
- Preheat H2O + Padlock probes then snap cool before adding Ampligase buffer and enzyme
- TB12kApr2016 V4: 423nM -> 4,978 probes -> ~85pM per probe
- Working concentration: 42.5pM per probe
- Add mix to sample and incubate for 30min at 37C
- Move sample to 60C and oven slowly decreases to 55C and held for ~24hr
Day 3
- Wash with 1X PBS once
- Add 1uM FISSEQ_ppRCA (1ul 200uM FISSEQ_ppRCA + 199ul 2X SSC + 30% formamide) and incubate 1hr at 55C
- Preheated to 70C before adding
- Wash with 2X SSC once, 1X SSC once, and 1X PBS once
- Add RCA mix and incubate 15hr at 30C
Component
|
Volume
|
H2O |
174
|
10X Phi29 Buffer |
20
|
25mM dNTP |
2
|
4mM aa-dUTP |
2
|
Phi 29 (low conc) |
2
|
Total |
200
|
Day 4
- Wash with 1X PBS once
- Add 200ul BS(PEG)9 (4ul BS(PEG)9 + 196ul 1X PBS) and incubate at RT for 1hr
- Wash with 1X PBS twice
- Add 1M Tris pH 8.0 and incubate at RT for 30min
- Wash with 1X PBS twice
Check Rolony
- Add 0.5uM FISGA_Adpt in 30% formamide + 2X SSC preheated to 75C
- Incubate for 10min at RT
- Wash with 2X SSC twice
- Image (saved in 7-11-2016)
Results
- Took images at 5 positions
File:20160706 BA8 DARTFISH MarkedPositions.jpg
Position 1
Position 2
Position 3
Position 4
Position 5