Matt:LabNotes/2016-8-3: Difference between revisions
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>Mzcai (Created page with "=DARTFISH on BA8 with and without PA Gel= *Try 3 samples with polyacrylamide gel added at different points in protocol *Sample 1: No gel *Sample 2: Gel added after permeabiliz...") |
>Mzcai |
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#*Secure in plastic jig | #*Secure in plastic jig | ||
#Add 50ul gel casting mix '''filtered and degassed''' | #Add 50ul gel casting mix '''filtered and degassed''' | ||
#*5% gel with | #*5% gel with 125uM Acrydite-Amine linker and 5mM BS(PEG)9 | ||
#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 | ||
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#Attach coverslip to bottom of petri dish | #Attach coverslip to bottom of petri dish | ||
#Aspirate non-polymerized gel and wash once with 1M Tris and once with cold 1X PBS | #Aspirate non-polymerized gel and wash once with 1M Tris and once with cold 1X PBS | ||
====Continue for All Samples==== | ====Continue for All Samples==== | ||
#Attach coverslip to bottom of petri dish | #Attach coverslip to bottom of petri dish |
Revision as of 22:11, 3 August 2016
DARTFISH on BA8 with and without PA Gel
- Try 3 samples with polyacrylamide gel added at different points in protocol
- Sample 1: No gel
- Sample 2: Gel added after permeabilization
- Sample 3: Gel added after reverse transcription
- Used 200nM padlock probe (twice the normal concentration)
- Gel mix and protocol is same as this with BS(PEG)9
Component | Volume |
40% AB 199:1 Mix | 6.25 |
10% BSA | 1 |
1mM Acryd/Amine Linker | 6.25 |
H2O | 33.5 |
5% TEMED | 1 |
5% APS | 1 |
BS(PEG)9 | 1 |
Total | 50 |
Protocol
Day 1
- Prepare 3 plastic culture dishes with 12x17mm hole, tweezers, large plastic dish, and 50C hot plate
- EtOH and UV sterilize
- Make 4% PFA in 1X PBS
- 10ml 16% PFA + 4ml 10X PBS + 26ml H2O
- Take out BA8 section from -80C and dry on 50C hot plate for 3min
- Submerge coverslip in 5ml 4% PFA in petri dish for 15min at 37C
- Wash twice with cold 1X SSPE by submerging
- 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
To Sample 2 Only
- Skip these steps and go to Reverse Transcription Immediately for other 2 samples
- Aspirate any liquid and place a glass slide with 50um spacer over the coverslip with tissue
- Secure in plastic jig
- Add 50ul gel casting mix filtered and degassed
- 5% gel with 125uM Acrydite-Amine linker and 5mM BS(PEG)9
- 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 1X PBS
- Attach coverslip to bottom of petri dish
- Aspirate non-polymerized gel and wash once with 1M Tris and once with cold 1X PBS
Continue for All Samples
- Attach coverslip to bottom of petri dish
- 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 FISSEQ_RT primer | 5 |
RNase Inhibitor | 2 |
M-MuLV RTase | 10 |
Total | 200 |
- Incubate 10min at 4C and then ~18hr at 37C