Matt:LabNotes/2016-2-29: Difference between revisions

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#Incubate for 10min at RT
#Incubate for 10min at RT
#Wash with 2X SSC twice
#Wash with 2X SSC twice
#Image (saved in 3-3-2016)
#Image (saved in 3-4-2016)
#*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 2mm from edge
#*Bottom of P wasn't covered by gel so image that as well
#Strip with 80% formamide preheated to 75C
#Strip with 80% formamide preheated to 75C
#Wash with 1X PBS twice
#Wash with 1X PBS twice
====0mm from edge====
[[File:MAX_20160229_BA8_DARTFISH_0mm_edge.jpg|450px]][[File:20160229_BA8_DARTFISH_0mm_edge_z24_ch01.jpg|450px]]
====2mm from edge====
[[File:MAX_20160229_BA8_DARTFISH_2mm_edge.jpg|450px]][[File:20160229_BA8_DARTFISH_2mm_edge_z24_ch01.jpg|450px]]
====4mm from edge====
[[File:MAX_20160229_BA8_DARTFISH_4mm_edge.jpg|450px]][[File:20160229_BA8_DARTFISH_4mm_edge_z21_ch01.jpg|450px]]
====6mm from edge====
[[File:MAX_20160229_BA8_DARTFISH_6mm_edge.jpg|450px]][[File:20160229_BA8_DARTFISH_6mm_edge_z22_ch01.jpg|450px]]
====8mm from edge====
[[File:MAX_20160229_BA8_DARTFISH_8mm_edge.jpg|450px]][[File:20160229_BA8_DARTFISH_8mm_edge_z17_ch01.jpg|450px]]
====bottom====
[[File:MAX_20160229_BA8_DARTFISH_bottom_nogel.jpg|450px]][[File:20160229_BA8_DARTFISH_bottom_nogel_z21_ch01.jpg|450px]]
===Conclusion===
*Compare with [[Matt:LabNotes/2016-1-20#Imaging_Results]]
*Tissue definitely had less degradation due to gel
*However also much fewer rolonies than with gel
*It is possible that gel prevented proper permeabilization and pepsin digestion since I did not change those times
**Pepsin digestion time especially may need to be increased
*Solution: Fix and pepsin digest BEFORE adding PA gel

Latest revision as of 01:22, 5 March 2016

DARTFISH on BA8 Sections with PA gel[edit]

not be below 6.5."

Protocol[edit]

Day 1[edit]

  1. Prepare plastic culture dish with 12x17mm hole, tweezers, large plastic dish, and 50C hot plate
    • EtOH and UV sterilize
  2. Used 4% PFA from 1/20/2016
  3. Take out second worst BA8 sections (second furthest front) 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. Aspirate any liquid and attach glass to bottom of culture dish with double-sided adhesive
  7. Add 100ul gel casting mix filtered and degassed
  8. Cover with plastic coverslips cut and glued together such that it leaves ~0.3mm space for gel height
  9. Seal in plastic bag and vacuum out air before filling with argon
  10. Let sit at RT for 30min
  11. Aspirate non-polymerized gel and wash once with cold 1X SSPE
    • Mostly did not polymerize
    • Only a ~1cm diameter circle of gel polymerized in center
    • I will continue with FISSEQ protocol to see whether can generate as many rolonies in gel area as non-gel area
  12. Add 0.25% TX-100 in 2XSSPE and incubate 10min at RT
  13. Wash with cold nf-H2O three times and check for degradation
  14. Add 0.01% Pepsin in 0.1N HCl and incubate for 90sec at 37C
    • 2ul 1%Pepsin + 10ul 2N HCl + 188ul H2O
  15. Wash with nf-1X PBS three times and check for degradation
  16. 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 must have expanded overnight and created a "tent" in the center of glass where gel is pushed up
    • The swelling is only on the grey matter (superficial layers) of tissue and nowhere else (white matter and glass) but could just be coincidence?
  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
  4. Add 1M Tris pH 8.0 and incubate 30min at RT
  5. Wash with 1X PBS twice
  6. RNA Removal
    • Add 200ul RNase Mix (20ul RNase H 10X Buffer + 2ul Riboshredder + 10ul RNase H + 168ul H2O) and incubate 1hr at 37C
  7. Wash with nf-H2O twice
  8. Prepare Ampligase mix on ice
    • Preheat H2O + Padlock probes + suppv2 Oligos to 85C and then snap cool before adding Ampligase buffer and enzyme
  • B13: 305nM
    • This batch of padlock probes was PCR'd an extra 3 cycles during production PCR
Component Volume
DEPC-H2O 18.43
Ampligase Buffer 10
Erin's Batch 13 2/12/2016 Padlock Probes 305nM]] 33
100nM suppv2 Oligos 28.57
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 ~20hr

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 18hr 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 300ul BS(PEG)9 (6ul BS(PEG)9 + 294ul 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 3-4-2016)
    • 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 2mm from edge
    • Bottom of P wasn't covered by gel so image that as well
  5. Strip with 80% formamide preheated to 75C
  6. Wash with 1X PBS twice

0mm from edge[edit]

File:MAX 20160229 BA8 DARTFISH 0mm edge.jpgFile:20160229 BA8 DARTFISH 0mm edge z24 ch01.jpg

2mm from edge[edit]

File:MAX 20160229 BA8 DARTFISH 2mm edge.jpgFile:20160229 BA8 DARTFISH 2mm edge z24 ch01.jpg

4mm from edge[edit]

File:MAX 20160229 BA8 DARTFISH 4mm edge.jpgFile:20160229 BA8 DARTFISH 4mm edge z21 ch01.jpg

6mm from edge[edit]

File:MAX 20160229 BA8 DARTFISH 6mm edge.jpgFile:20160229 BA8 DARTFISH 6mm edge z22 ch01.jpg

8mm from edge[edit]

File:MAX 20160229 BA8 DARTFISH 8mm edge.jpgFile:20160229 BA8 DARTFISH 8mm edge z17 ch01.jpg

bottom[edit]

File:MAX 20160229 BA8 DARTFISH bottom nogel.jpgFile:20160229 BA8 DARTFISH bottom nogel z21 ch01.jpg

Conclusion[edit]

  • Compare with Matt:LabNotes/2016-1-20#Imaging_Results
  • Tissue definitely had less degradation due to gel
  • However also much fewer rolonies than with gel
  • It is possible that gel prevented proper permeabilization and pepsin digestion since I did not change those times
    • Pepsin digestion time especially may need to be increased
  • Solution: Fix and pepsin digest BEFORE adding PA gel