Matt:LabNotes/2016-3-14: Difference between revisions
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>Mzcai m (→Day 1) |
>Mzcai m (→Check Rolony) |
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(6 intermediate revisions by the same user not shown) | |||
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| Total||200 | | Total||200 | ||
|} | |} | ||
#Incubate 10min at 4C and then ~18hr at 37C | #Incubate 10min at 4C and then ~18hr at 37C | ||
===Day 2=== | ===Day 2=== | ||
#Wash with 1X PBS once | #Wash with 1X PBS once | ||
#Add 200ul cold BS(PEG)9 (4ul stock BS(PEG)9 + 196ul 1X PBS) and incubate 1hr at RT | #Add 200ul cold BS(PEG)9 (4ul stock BS(PEG)9 + 196ul 1X PBS) and incubate 1hr at RT | ||
Line 66: | Line 64: | ||
#Prepare Ampligase mix on ice | #Prepare Ampligase mix on ice | ||
#*Preheat H2O + Padlock probes + suppv2 Oligos to 85C and then snap cool before adding Ampligase buffer and enzyme | #*Preheat H2O + Padlock probes + suppv2 Oligos to 85C and then snap cool before adding Ampligase buffer and enzyme | ||
* | *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|| | | DEPC-H2O||5.1 | ||
|- | |- | ||
| Ampligase Buffer||10 | | Ampligase Buffer||10 | ||
|- | |- | ||
| | | Justin's Batch 9 9/11/2015 Padlock Probes 216nM]]||46.3 | ||
|- | |- | ||
| 100nM suppv2 Oligos || 28.57 | | 100nM suppv2 Oligos || 28.57 | ||
Line 85: | Line 82: | ||
|} | |} | ||
#Add mix to sample and incubate for 30min at 37C | #Add mix to sample and incubate for 30min at 37C | ||
#Move sample to 60C and oven slowly decreases to 55C and held for ~ | #Move sample to 60C and oven slowly decreases to 55C and held for ~24hr | ||
===Day 3=== | ===Day 3=== | ||
*Center of gel was not covered with liquid when taking out | |||
#Wash with 1X PBS once | #Wash with 1X PBS once | ||
#Add 1uM FISSEQ_ppRCA (1ul 200uM FISSEQ_ppRCA + 199ul 2X SSC + 30% formamide) and incubate 1hr at 55C | #Add 1uM FISSEQ_ppRCA (1ul 200uM FISSEQ_ppRCA + 199ul 2X SSC + 30% formamide) and incubate 1hr at 55C | ||
#*Preheated to 70C before adding | #*Preheated to 70C before adding | ||
#Wash with 2X SSC once, 1X SSC once, and 1X PBS once | #Wash with 2X SSC once, 1X SSC once, and 1X PBS once | ||
#Add RCA mix and incubate | #Add RCA mix and incubate 15hr at 30C | ||
{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''Component''' | | align="center" style="background:#f0f0f0;"|'''Component''' | ||
Line 119: | Line 117: | ||
#Incubate for 10min at RT | #Incubate for 10min at RT | ||
#Wash with 2X SSC twice | #Wash with 2X SSC twice | ||
#Image (saved in 3- | #Image (saved in 3-18-2016) | ||
#*As usual have the tissue oriented like a backwards 'P' | #*As usual have the tissue oriented like a backwards 'P' | ||
#*Image on horizontal line at widest section of 'P' | #*Image on horizontal line at widest section of 'P' | ||
#**Edge is right side of backwards 'P' (ie the vertical edge) | #**Edge is right side of backwards 'P' (ie the vertical edge) | ||
#**Take images every | #**Take images every certain distance from edge | ||
#*Bottom of P wasn't covered by gel so image that as well | #*Bottom of P wasn't covered by gel so image that as well | ||
==== | ===Results=== | ||
*Images not shown because no rolonies anywhere | |||
*Only some autofluorescence or non-specific fluorescence | |||
===Conclusion=== | ===Conclusion=== | ||
* | *My guess is that the 4% PFA in hydrogel solution over cross-linked RNA during gel polymerization? | ||
*This prevents reverse transcription | |||
* | |||
Latest revision as of 20:16, 18 March 2016
DARTFISH on BA8 Sections with PA gel[edit]
- Last time using gel to cover section it swelled during RT incubation
- To prevent swelling going to use 4% formaldehyde in the gel solution, which crosslinks the hydrogel to Vectabond treated glass (amine functional groups)
- Also add hydrogel after permeabilization because I think last time the hydrogel prevented effective permeabilization
- Used 4th best section from 1/19/16 BA8 patient 1568 from Yun
- 4% Formaldehyde for fixation is one week old and has been sitting at RT under argon gas
- 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[edit]
Day 1[edit]
- Prepare plastic culture dish with 12x17mm hole, tweezers, large plastic dish, and 50C hot plate
- EtOH and UV sterilize
- Used 4% PFA from 3/8/2016
- Take out second best remaining BA8 sections (4th from back) 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 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
- Add 75ul gel casting mix filtered and degassed
- 4% PFA, 10% Acrylamide/Bis-Acrylamide (199:1), 0.2% BSA, 0.1% APS, 0.1% TEMED
- Cover with plastic coverslips cut and glued together such that it leaves ~0.25mm 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
- Edges did not polymerize but the grey matter is fully covered
- 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[edit]
- 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
- B9: 216nM
Component | Volume |
DEPC-H2O | 5.1 |
Ampligase Buffer | 10 |
Justin's Batch 9 9/11/2015 Padlock Probes 216nM]] | 46.3 |
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 ~24hr
Day 3[edit]
- Center of gel was not covered with liquid when taking out
- 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 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]
- 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[edit]
- 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 3-18-2016)
- 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 certain distance from edge
- Bottom of P wasn't covered by gel so image that as well
Results[edit]
- Images not shown because no rolonies anywhere
- Only some autofluorescence or non-specific fluorescence
Conclusion[edit]
- My guess is that the 4% PFA in hydrogel solution over cross-linked RNA during gel polymerization?
- This prevents reverse transcription