Matt:LabNotes/2016-6-29: Difference between revisions

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*Used worst section from [[Matt:LabNotes/2016-1-20|1/19/16 BA8 patient 1568 from Yun]]
*Used worst section from [[Matt:LabNotes/2016-1-20|1/19/16 BA8 patient 1568 from Yun]]
*These coverslips have only Vectabond (-NH2 primary amines) and no Bind-Silane


*Gel mix and protocol is same as [[Matt:LabNotes/2016-2-18|this successful experiment]]
*Gel mix and protocol is same as [[Matt:LabNotes/2016-2-18|this successful experiment]]
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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
#Aspirate non-polymerized gel and wash once with 1X PBS
#Attach coverslip to bottom of petri dish
#Attach coverslip to bottom of petri dish
#Prepare Reverse Transcription Mix '''on ice''' and add
#*COVERSLIP CRACKED DIAGONALLY WHEN CLEANING AWAY GEL
{| {{table}}
#*NEED TO BE MORE CAREFUL WHEN HANDLING
| align="center" style="background:#f0f0f0;"|'''Components'''
*Continued by testing for swelling
| align="center" style="background:#f0f0f0;"|'''Volume'''
#Put each half of cracked coverslip in petri dish with 1M Tris and put in 37C overnight
|-
| 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 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?
#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 + 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
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Component'''
| align="center" style="background:#f0f0f0;"|'''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
|}
#Add mix to sample and incubate for 30min at 37C
#Move sample to 60C and oven slowly decreases to 55C and held for ~20hr
===Day 3===
#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 18hr 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===
===Results===
#Add 0.5uM FISGA_Adpt in 30% formamide + 2X SSC preheated to 75C
[[File:20160630_GelTissueSwellTest_1.JPG|450px]]
#Incubate for 10min at RT
[[File:20160630_GelTissueSwellTest_2.JPG|450px]]
#Wash with 2X SSC twice
#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
#Wash with 1X PBS twice


====0mm from edge====
*Next morning the gels were still stuck to glass
[[File:MAX_20160229_BA8_DARTFISH_0mm_edge.jpg|450px]][[File:20160229_BA8_DARTFISH_0mm_edge_z24_ch01.jpg|450px]]
*No significant swelling
====2mm from edge====
**Vertical length of gel was ~17mm when cast (no image but that is the length of the mold) and is ~17mm after overnight incubation in Tris
[[File:MAX_20160229_BA8_DARTFISH_2mm_edge.jpg|450px]][[File:20160229_BA8_DARTFISH_2mm_edge_z24_ch01.jpg|450px]]
**Suggests that the gel is covalently bound to glass
====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===
===Conclusion===
*Compare with [[Matt:LabNotes/2016-1-20#Imaging_Results]]
*Making gel with Acrydite/Amine linker and BS(PEG)9 is viable solution to covalently bond polyacrylamide gel to Vectabond treated glass and tissue sections
*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 00:39, 1 July 2016

FISSEQ on BA8 Sections with PA gel + Acrydite/BS(PEG)9[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 6/15/2016
  3. Take out worst BA8 section that is left 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 120um spacer over the coverslip with tissue
    • Secure in plastic jig
  11. Add 100ul 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
    • COVERSLIP CRACKED DIAGONALLY WHEN CLEANING AWAY GEL
    • NEED TO BE MORE CAREFUL WHEN HANDLING
  • Continued by testing for swelling
  1. Put each half of cracked coverslip in petri dish with 1M Tris and put in 37C overnight

Results[edit]

File:20160630 GelTissueSwellTest 1.JPG File:20160630 GelTissueSwellTest 2.JPG

  • Next morning the gels were still stuck to glass
  • No significant swelling
    • Vertical length of gel was ~17mm when cast (no image but that is the length of the mold) and is ~17mm after overnight incubation in Tris
    • Suggests that the gel is covalently bound to glass

Conclusion[edit]

  • Making gel with Acrydite/Amine linker and BS(PEG)9 is viable solution to covalently bond polyacrylamide gel to Vectabond treated glass and tissue sections