Matt:LabNotes/2016-8-3: Difference between revisions
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*Sample 1: No gel | *Sample 1: No gel | ||
*Sample 2: Gel added after permeabilization | *Sample 2: Gel added after permeabilization | ||
*Sample 3: Gel added after | *Sample 3: Gel added after RNA removal | ||
*Used 200nM padlock probe (twice the normal concentration) | *Used 200nM padlock probe (twice the normal concentration) | ||
Line 94: | Line 94: | ||
| Total||200 | | Total||200 | ||
|} | |} | ||
#Incubate 10min at 4C and then ~ | #Incubate 10min at 4C and then ~15hr at 37C | ||
===Day 2=== | ===Day 2=== | ||
#Wash with 1X PBS once | #Wash with 1X PBS once | ||
Line 105: | Line 105: | ||
#*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 | ||
#Add mix to sample and incubate for | ====To Sample 3 Only==== | ||
#Add 50ul gel casting mix '''filtered and degassed''' | |||
#*5% gel with 125uM Acrydite-Amine linker, 5mM BS(PEG)9, and Use [[Matt:LabNotes/2016-7-26#Qubit_ssDNA | 478nM TB12kApr2016 V4 Probes]] instead of H2O (33.5ul) | |||
#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 1M Tris | |||
#Prepare Ampligase mix on ice | |||
#*Preheat Padlock Probes then snap cool before adding Ampligase buffer and enzyme | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Component''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume''' | |||
|- | |||
| Ampligase Buffer||10 | |||
|- | |||
| [[Matt:LabNotes/2016-7-26#Qubit_ssDNA | 478nM]]||80 | |||
|- | |||
| 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 | |||
====To Samples 1 & 2==== | |||
#Prepare Ampligase mix on ice | |||
#*Preheat Padlock Probes then snap cool before adding Ampligase buffer and enzyme | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Component''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume''' | |||
|- | |||
| Ampligase Buffer||10 | |||
|- | |||
| [[Matt:LabNotes/2016-7-26#Qubit_ssDNA | 478nM]]||80 | |||
|- | |||
| 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=== | |||
#Wash with 1X PBS twice | #Wash with 1X PBS twice | ||
#Add 200ul (1ul 200uM FISSEQ_RCA + 199ul 2X SSC + 30% formamide) and incubate 1hr at 60C | #Add 200ul (1ul 200uM FISSEQ_RCA + 199ul 2X SSC + 30% formamide) and incubate 1hr at 60C | ||
Line 130: | Line 171: | ||
#Add RCA mix and incubate at 30C overnight (~15hrs) | #Add RCA mix and incubate at 30C overnight (~15hrs) | ||
===Day | ===Day 4=== | ||
#Wash with 1X PBS once | #Wash with 1X PBS once | ||
#Add 200ul BS(PEG)9 (4ul BS(PEG)9 + 196ul 1X PBS) and incubate at RT for 1hr | #Add 200ul BS(PEG)9 (4ul BS(PEG)9 + 196ul 1X PBS) and incubate at RT for 1hr |
Revision as of 21:28, 4 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 RNA removal
- 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 ~15hr at 37C
Day 2
- 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
To Sample 3 Only
- Add 50ul gel casting mix filtered and degassed
- 5% gel with 125uM Acrydite-Amine linker, 5mM BS(PEG)9, and Use 478nM TB12kApr2016 V4 Probes instead of H2O (33.5ul)
- 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 1M Tris
- Prepare Ampligase mix on ice
- Preheat Padlock Probes then snap cool before adding Ampligase buffer and enzyme
Component | Volume |
Ampligase Buffer | 10 |
478nM | 80 |
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
To Samples 1 & 2
- Prepare Ampligase mix on ice
- Preheat Padlock Probes then snap cool before adding Ampligase buffer and enzyme
Component | Volume |
Ampligase Buffer | 10 |
478nM | 80 |
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