Matt:LabNotes/2016-7-6: Difference between revisions
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>Mzcai (Created page with "=DARTFISH on BA8 sections with PA gel + Acrydite/BS(PEG)9= *[[Matt:LabNotes/2016-7-6|Yesterday tried this twice but first time tissue degraded and second time dish leaked duri...") |
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#Incubate 10min at 4C and then ~18hr at 37C | #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 | |||
*[[Picture]] | |||
#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 | |||
*B9: 216nM | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Component''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume''' | |||
|- | |||
| DEPC-H2O||5.1 | |||
|- | |||
| Ampligase Buffer||10 | |||
|- | |||
| [[TB12k V4 Padlock Probes 216nM]]||46.3 | |||
|- | |||
| Ampligase||10 | |||
|- | |||
| Total||100 | |||
|} | |||
#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=== | |||
*Center of gel was not covered with liquid when taking out | |||
#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 | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Component''' | |||
| align="center" style="background:#f0f0f0;"|'''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 300ul BS(PEG)9 (6ul BS(PEG)9 + 294ul 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 3-18-2016) | |||
#*As usual have the tissue oriented like a backwards 'P' | |||
#*Image on horizontal line at widest section of 'P' | |||
#**Edge is right side of backwards 'P' (ie the vertical edge) | |||
#**Take images every certain distance from edge | |||
#*Bottom of P wasn't covered by gel so image that as well--> |
Revision as of 19:37, 7 July 2016
DARTFISH on BA8 sections with PA gel + Acrydite/BS(PEG)9
- Gel mix and protocol is same as this with 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
- EtOH and UV sterilize
- 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
- Secure in plastic jig
- 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