Matt:LabNotes/2016-2-23: Difference between revisions

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>Mzcai
>Mzcai
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#Seal in plastic bag and vacuum out air before filling with argon
#Seal in plastic bag and vacuum out air before filling with argon
#Let sit at RT for 30min
#Let sit at RT for 30min
#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
#Add 0.25% TX-100 in 2XSSPE and incubate 10min at RT
#Add 0.25% TX-100 in 2XSSPE and incubate 10min at RT
#Wash with cold nf-H2O three times '''and check for degradation'''
#Wash with cold nf-H2O three times '''and check for degradation'''
Line 52: Line 56:
| Total||200
| Total||200
|}
|}
#Incubate 10min at 4C and then ~18hr at 37C<!--
#Incubate 10min at 4C and then ~15hr at 37C
===Day 2===
===Day 2===
#Wash with 1X PBS once
#Wash with 1X PBS once
Line 59: Line 63:
#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
#*At the maximum width, width = 8mm. The border from 7-8mm is very thin
#RNA Removal
#RNA Removal
#*Add 200ul RNase Mix (20ul RNase H 10X Buffer + 2ul Riboshredder + 10ul RNase H + 168ul H2O) and incubate 1hr at 37C
#*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
#Wash with nf-H2O twice
#Prepare Ampligase mix on ice
#Prepare FISSEQ mix on ice
#*Preheat H2O + Padlock probes + suppv2 Oligos to 85C and then snap cool before adding Ampligase buffer and enzyme
#Add 200ul CircLigaseII mix and incuabate 3hr at 60C
*B9: 216nM
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Component'''
| align="center" style="background:#f0f0f0;"|'''Component'''
| align="center" style="background:#f0f0f0;"|'''Volume'''
| align="center" style="background:#f0f0f0;"|'''Volume'''
|-
|-
| DEPC-H2O||17.43
| DEPC-H2O||640
|-
|-
| Ampligase Buffer||10
| CircLigase Buffer 10X||100
|-
|-
| Justin's Batch 8 7/22/2015 Padlock Probes 298nM]]||34
| MnCl2 50mM||50
|-
|-
| 100nM suppv2 Oligos || 28.57
| Betaine 5M|| 200
|-
|-
| Ampligase||10
| CircLigase II (100U/ul)||10
|-
|-
| Total||100
| Total||1000
|}
|}
#Wash with 1X PBS twice
#Add 200ul 0.5uM FISSEQ_RCA (5ul 100uM FISSEQ_RCA + 995ul 2X SSC + 30% formamide) and incubate 1hr at 60C
#Wash with 2X SSC + 30% formamide for 10min at 60C
#wash with 2X SSC, 1X SSC, 1X PBS once each
#Add 200ul RCA mix and incubate for 15hr at 30C
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Component'''
| align="center" style="background:#f0f0f0;"|'''Component'''
| align="center" style="background:#f0f0f0;"|'''Volume'''
| align="center" style="background:#f0f0f0;"|'''Volume'''
|-
|-
| DEPC-H2O||5.43
| H2O||174
|-
|-
| Ampligase Buffer||10
| 10X Phi29 Buffer||20
|-
|-
| Justin's Batch 9 7/22/2015 Padlock Probes 216nM]]||26
| 25mM dNTP||2
|-
|-
| 100nM suppv2 Oligos || 28.57
| 4mM aa-dUTP||2
|-
|-
| Ampligase||10
| Phi 29 (low conc)||2
|-
|-
| Total||100
| Total||200
|}
|}
#Add mix to sample and incubate 30min at 37C
#Move to 60C as oven slowly decreases to 55C and held for another ~18hr


===Day 3===
===Day 3===

Revision as of 21:54, 24 February 2016

DARTFISH on BA8 Sections with PA gel

3 months after they were initially mixed. The solution should be clear, colorless, with no precipitate and the pH should not be below 6.5."

Protocol

Day 1

  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 worst BA8 sections (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 0.5mm space for gel height
    • FORGOT TO DO THIS SO GEL WON'T BE FLAT ON TOP
  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 ~15hr at 37C

Day 2

  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 FISSEQ mix on ice
  9. Add 200ul CircLigaseII mix and incuabate 3hr at 60C
Component Volume
DEPC-H2O 640
CircLigase Buffer 10X 100
MnCl2 50mM 50
Betaine 5M 200
CircLigase II (100U/ul) 10
Total 1000
  1. Wash with 1X PBS twice
  2. Add 200ul 0.5uM FISSEQ_RCA (5ul 100uM FISSEQ_RCA + 995ul 2X SSC + 30% formamide) and incubate 1hr at 60C
  3. Wash with 2X SSC + 30% formamide for 10min at 60C
  4. wash with 2X SSC, 1X SSC, 1X PBS once each
  5. Add 200ul RCA mix and incubate for 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 3

  1. Wash with 1X PBS once
  2. To DARTFISH slide 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
    • Washed with 1X PBS another time and let sit in PBS for ~10min at RT because the RCA mix partially froze in the chilled tube holder so I had to remake the RCA mix
  4. Add RCA mix and incubate 10.5hr at 30C
Component Volume
H2O 174
10X Phi29 Buffer 20
25mM dNTP 2
4mM aa-dUTP 2
Phi 29 (low conc) 2
Total 200
  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

  1. Add 0.5uM FISGA_Adpt in 2X SSC preheated to 75C
  2. Incubate for 10min at RT
  3. Wash with 2X SSC twice
  4. Image (saved in 1-26-2015)
  5. Strip with 80% formamide preheated to 75C
  6. Wash with 1X PBS twice


Imaging Results

  • Imaged 1-26-2016
  • 63X with 0.3um step size
  • 2048x2048
  • Gain = 600 Offset = 1%

Sample A

White matter edge

  • Edge of tissue on vertical side

File:MAX BA8 A white edge.jpgFile:BA8 A white edge z24 ch01.jpg

2mm from edge

File:MAX BA8 A 2mm from edge.jpgFile:BA8 A 2mm from edge z18 ch01.jpg

4mm from edge

File:MAX BA8 A 4mm from edge.jpgFile:BA8 A 4mm from edge z21 ch01.jpg

6mm from edge

File:MAX BA8 A 6mm from edge.jpgFile:BA8 A 6mm from edge z15 ch01.jpg

Sample B

White matter edge

  • Edge of tissue on vertical side

File:MAX BA8 B white edge.jpgFile:BA8 B white edge z15 ch01.jpg

2mm from edge

File:MAX BA8 B 2mm from edge.jpgFile:BA8 B 2mm from edge z15 ch01.jpg

4mm from edge

File:MAX BA8 B 4mm from edge.jpgFile:BA8 B 4mm from edge z15 ch01.jpg

Conclusion

  • Even with this minimal pepsin digestion and careful washing there's too much tissue degradation
  • Problem is compounded by the fact that the most interesting area (grey matter with cortical layers) is also the most fragile
  • Need to try adding polyacrylamide gel layer over the tissue to prevent tissue degradation

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