Matt:LabNotes/2016-7-6: Difference between revisions
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#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:20160706_BA8_Hydrogel_Day2.JPG|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 118: | Line 118: | ||
#Add mix to sample and incubate for 30min at 37C | #Add mix to sample and incubate for 30min at 37C | ||
#Move sample to 60C and oven slowly decreases to 55C and held for ~24hr | #Move sample to 60C and oven slowly decreases to 55C and held for ~24hr | ||
===Day 3=== | ===Day 3=== | ||
#Wash with 1X PBS once | #Wash with 1X PBS once | ||
Line 152: | Line 153: | ||
#Wash with 2X SSC twice | #Wash with 2X SSC twice | ||
#Image (saved in 7-11-2016) | #Image (saved in 7-11-2016) | ||
==Results== | |||
*Took images at 5 positions | |||
[[File:20160706_BA8_DARTFISH_MarkedPositions.jpg|450px]] | |||
===Position 1=== | |||
[[File:MAX_20160706_FISGAadpt_Pos1_bottom_.jpg|350px]][[File:20160706_FISGAadpt_Pos1_bottom_z09_ch01.jpg|350px]] | |||
===Position 2=== | |||
[[File:MAX_20160706_FISGAadpt_Pos2_topleft_.jpg|350px]][[File:20160706_FISGAadpt_Pos2_topleft_z03_ch01.jpg|350px]] | |||
===Position 3=== | |||
[[File:MAX_20160706_FISGAadpt_Pos3_topleft_.jpg|350px]][[File:20160706_FISGAadpt_Pos3_topleft_z02_ch01.jpg|350px]] | |||
===Position 4=== | |||
[[File:MAX_20160706_FISGAadpt_Pos4_topmid_.jpg|350px]][[File:20160706_FISGAadpt_Pos4_topmid_z00_ch01.jpg|350px]] | |||
===Position 5=== | |||
[[File:MAX_20160706_FISGAadpt_Pos5_bottom_.jpg|350px]][[File:20160706_FISGAadpt_Pos5_bottom_z02_ch01.jpg|350px]] | |||
==Conclusion== | |||
*Gel keeps tissue intact and doesn't totally inhibit DARTFISH | |||
*Decent number of rolonies in white matter but not as many as best time with CA12kNov2014 V4 probeset | |||
**This is a different probeset and first time using it in tissue so can't directly compare | |||
**The CA12kNov2014 V4 probeset had some probes targeting repeat regions that made up a significant fraction of the rolonies | |||
*Need to cover the gray matter with gel and see how many rolonies when it is not degraded |
Latest revision as of 23:14, 25 July 2016
DARTFISH on BA8 sections with PA gel + Acrydite/BS(PEG)9[edit]
- 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[edit]
Day 1[edit]
- 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[edit]
- 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
Component | Volume |
DEPC-H2O | 30 |
Ampligase Buffer | 10 |
TB12k V4 Padlock Probes 423nM | 50 |
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[edit]
- 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[edit]
- 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[edit]
- 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[edit]
- Took images at 5 positions
File:20160706 BA8 DARTFISH MarkedPositions.jpg
Position 1[edit]
File:MAX 20160706 FISGAadpt Pos1 bottom .jpgFile:20160706 FISGAadpt Pos1 bottom z09 ch01.jpg
Position 2[edit]
File:MAX 20160706 FISGAadpt Pos2 topleft .jpgFile:20160706 FISGAadpt Pos2 topleft z03 ch01.jpg
Position 3[edit]
File:MAX 20160706 FISGAadpt Pos3 topleft .jpgFile:20160706 FISGAadpt Pos3 topleft z02 ch01.jpg
Position 4[edit]
File:MAX 20160706 FISGAadpt Pos4 topmid .jpgFile:20160706 FISGAadpt Pos4 topmid z00 ch01.jpg
Position 5[edit]
File:MAX 20160706 FISGAadpt Pos5 bottom .jpgFile:20160706 FISGAadpt Pos5 bottom z02 ch01.jpg
Conclusion[edit]
- Gel keeps tissue intact and doesn't totally inhibit DARTFISH
- Decent number of rolonies in white matter but not as many as best time with CA12kNov2014 V4 probeset
- This is a different probeset and first time using it in tissue so can't directly compare
- The CA12kNov2014 V4 probeset had some probes targeting repeat regions that made up a significant fraction of the rolonies
- Need to cover the gray matter with gel and see how many rolonies when it is not degraded