Matt:LabNotes/2016-7-6: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Mzcai
>Mzcai
mNo edit summary
 
(2 intermediate revisions by the same user not shown)
Line 153: 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]

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]

  1. Prepare two plastic culture dishes with 12x17mm hole, tweezers, large plastic dish, and 50C hot plate
    • EtOH and UV sterilize
  2. Make fresh 4% PFA in 1X PBS
  3. Take out BA8 section from -80C and dry on 50C hot plate for 3min
  4. Submerge coverslip in 5ml 4% PFA in 6 well culture plate for 15min at 37C
  5. Wash twice with cold 1X SSPE by submerging
  6. Add 0.25% TX-100 in 2XSSPE and incubate 10min at RT
  7. Wash with cold nf-H2O three times and check for degradation
  8. Add 0.01% Pepsin in 0.1N HCl and incubate for 90sec at 37C
    • 2ul 1%Pepsin + 10ul 2N HCl + 188ul H2O
  9. Wash with nf-1X PBS three times and check for degradation
  10. Aspirate any liquid and place a glass slide with 50um spacer over the coverslip with tissue
    • Secure in plastic jig
  11. Add 50ul gel casting mix filtered and degassed
    • 5% gel with 500uM Acrydite-Amine linker and 5mM BS(PEG)9
  12. Seal in plastic bag and vacuum out air before filling with argon
  13. Let sit at RT for 30min
  14. Aspirate non-polymerized gel and wash once with 1X PBS
  15. Attach coverslip to bottom of petri dish
  16. Aspirate non-polymerized gel and wash once with 1M Tris and once with cold 1X SSPE
  17. 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
  1. Incubate 10min at 4C and then ~18hr at 37C

Day 2[edit]

  • Gel does not look significantly swollen
  1. Wash with 1X PBS once
  2. Add 200ul cold BS(PEG)9 (4ul stock BS(PEG)9 + 196ul 1X PBS) and incubate 1hr at RT
  3. Wash with 1X PBS twice
  1. Add 1M Tris pH 8.0 and incubate 30min at RT
  2. Wash with 1X PBS twice
  3. RNA Removal
    • Add 200ul RNase Mix (20ul RNase H 10X Buffer + 2ul Riboshredder + 10ul RNase H + 168ul H2O) and incubate 1hr at 37C
  4. Wash with nf-H2O twice
  5. 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
  1. Add mix to sample and incubate for 30min at 37C
  2. Move sample to 60C and oven slowly decreases to 55C and held for ~24hr

Day 3[edit]

  1. Wash with 1X PBS once
  2. Add 1uM FISSEQ_ppRCA (1ul 200uM FISSEQ_ppRCA + 199ul 2X SSC + 30% formamide) and incubate 1hr at 55C
    • Preheated to 70C before adding
  3. Wash with 2X SSC once, 1X SSC once, and 1X PBS once
  4. 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]

  1. Wash with 1X PBS once
  2. Add 200ul BS(PEG)9 (4ul BS(PEG)9 + 196ul 1X PBS) and incubate at RT for 1hr
  3. Wash with 1X PBS twice
  4. Add 1M Tris pH 8.0 and incubate at RT for 30min
  5. Wash with 1X PBS twice

Check Rolony[edit]

  1. Add 0.5uM FISGA_Adpt in 30% formamide + 2X SSC preheated to 75C
  2. Incubate for 10min at RT
  3. Wash with 2X SSC twice
  4. 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