Matt:LabNotes/2016-2-29: Difference between revisions
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>Mzcai m (→Check Rolony) |
>Mzcai m (→Conclusion) |
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*It is possible that gel prevented proper permeabilization and pepsin digestion since I did not change those times | *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 | **Pepsin digestion time especially may need to be increased | ||
*Solution: Fix and pepsin digest BEFORE adding PA gel |
Revision as of 01:18, 5 March 2016
DARTFISH on BA8 Sections with PA gel
- Used worst section from 1/19/16 BA8 patient 1568 from Yun
- Gel mix and protocol is same as this successful experiment
- 4% Formaldehyde is one month old and has been sitting at RT
- From <http://swehsc.pharmacy.arizona.edu/sites/swehsc.pharmacy.arizona.edu/files/docs/ci/formaldehyde.pdf>
- "We recommend that 10% buffered formalin solutions be used no longer than 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
- Prepare plastic culture dish with 12x17mm hole, tweezers, large plastic dish, and 50C hot plate
- EtOH and UV sterilize
- Used 4% PFA from 1/20/2016
- Take out second worst BA8 sections (second furthest front) 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
- Aspirate any liquid and attach glass to bottom of culture dish with double-sided adhesive
- Add 100ul gel casting mix filtered and degassed
- Cover with plastic coverslips cut and glued together such that it leaves ~0.3mm space for gel height
- 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 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
- 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
- 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
- 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
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 |
- 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
Component | 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
- 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 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
File:MAX 20160229 BA8 DARTFISH 0mm edge.jpgFile:20160229 BA8 DARTFISH 0mm edge z24 ch01.jpg
2mm from edge
File:MAX 20160229 BA8 DARTFISH 2mm edge.jpgFile:20160229 BA8 DARTFISH 2mm edge z24 ch01.jpg
4mm from edge
File:MAX 20160229 BA8 DARTFISH 4mm edge.jpgFile:20160229 BA8 DARTFISH 4mm edge z21 ch01.jpg
6mm from edge
File:MAX 20160229 BA8 DARTFISH 6mm edge.jpgFile:20160229 BA8 DARTFISH 6mm edge z22 ch01.jpg
8mm from edge
File:MAX 20160229 BA8 DARTFISH 8mm edge.jpgFile:20160229 BA8 DARTFISH 8mm edge z17 ch01.jpg
bottom
File:MAX 20160229 BA8 DARTFISH bottom nogel.jpgFile:20160229 BA8 DARTFISH bottom nogel z21 ch01.jpg
Conclusion
- 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