Matt:LabNotes/2015-8-18: Difference between revisions
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| Total||200 | | Total||200 | ||
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#Incubate 10min at 4C and then ~18hr at 37C | #Incubate 10min at 4C and then ~18hr at 37C | ||
#Wash with 1X PBS once | #Wash with 1X PBS once | ||
#Add 300ul BS(PEG)9 (6ul BS(PEG)9 + 294ul 1X PBS) and incubate 1hr at RT | #Add 300ul BS(PEG)9 (6ul BS(PEG)9 + 294ul 1X PBS) and incubate 1hr at RT | ||
Line 52: | Line 52: | ||
#RNA Removal | #RNA Removal | ||
#*Add RNase Mix (20ul RNase H 10X Buffer + 2ul Riboshredder + 10ul RNase H + 168ul H2O) and incubate 1hr at 37C | #*Add RNase Mix (20ul RNase H 10X Buffer + 2ul Riboshredder + 10ul RNase H + 168ul H2O) and incubate 1hr at 37C | ||
#Wash with H2O twice | |||
#Wash with | #Add 100nM Padlock Probe Mix with 1000X suppv2 oligos and incubate ~1hr at 60C as oven slowly decreases to 55C and held for another ~20hr | ||
#*Wrapped with parafilm to prevent evaporation | |||
#Add | |||
#*Wrapped with parafilm to prevent evaporation | |||
{| {{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||20.18 | ||
|- | |- | ||
| Ampligase Buffer|| | | Ampligase Buffer||10 | ||
|- | |- | ||
| [[Matt:JustinLabNotes/2015-7- | | [[Matt:JustinLabNotes/2015-7-22#Qubit_Quantification | Padlock Probes 320nM]]||31.25 | ||
|- | |- | ||
| | | 100nM suppv2 Oligos || 28.57 | ||
|- | |- | ||
| Total|| | | Ampligase||10 | ||
|- | |||
| Total||100 | |||
|} | |} | ||
#Wash with 1X PBS once | #Wash with 1X PBS once | ||
#Add 1uM FISSEQ_ppRCA ( | #Add 1uM FISSEQ_ppRCA (1ul 200uM FISSEQ_ppRCA + 199ul 2X SSC + 30% formamide) and incubate 1hr at 55C | ||
#*Preheated to | #*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 6hr at 30C | #Add RCA mix and incubate 6hr at 30C |
Revision as of 18:06, 20 August 2015
BA8 Section DARTFISH suppv2 with Fiducial Beads
- Repeat Matt:LabNotes/2015-8-17 but with fresh PFA and 0.01N HCl (instead of 0.1N during pepsin step) to see if that helps with tissue degradation
- 3-4 month old BA8 tissue section
- suppv2 design
- Use FluoSpheres Amine-Modified Microspheres 0.2um (505/515) as fiducial markers
- Try only 5min incubation with TX-500 to see if that decreases tissue degradation
DARTFISH on BA8
- Prepare plastic culture dish with hole, tweezers, large plastic dish, and 50C hot plate
- EtOH sterilize and UV
- Made 40ml 4% PFA at RT
- 10ml 16% PFA from ampule + 4ml 10X PBS + 26ml nuclease-free H2O
- Take out BA8 section from -80C and incubate on 50C hot plate for 3min
- Submerge section in 5ml 4% PFA in 6 well culture plate for 15min at 37C
- Wash with 2X SSPE by submerging in 2 clean wells of culture plate with 5ml SSPE
- Aspirate any liquid and attach glass to bottom of culture dish with double-sided adhesive
- Add 0.25% TX-100 in 2XSSPE and incubate 5min at RT
- New solution made by Justin
- Wash with nuclease-free H2O three times
- Add 0.01% Pepsin in 0.1N HCl and incubate for 5min at 37C
- 2ul 1%Pepsin + 1ul 2N HCl + 197ul H2O
- Wash with nuclease-free PBS three times
- Prepare Reverse Transcription Mix on ice
Components | Volume |
H2O | 157 |
10X M-MuLV Buffer | 20 |
25mM dNTP | 2 |
2mM aa-dUTP | 4 |
100uM N9 | 5 |
RNase Inhibitor | 2 |
M-MuLV RTase | 10 |
Total | 200 |
- Incubate 10min at 4C and then ~18hr at 37C
- Wash with 1X PBS once
- Add 300ul BS(PEG)9 (6ul BS(PEG)9 + 294ul 1X PBS) and incubate 1hr at RT
- Wash with 1X PBS twice
- Add Tris pH8.0 and incubate 30min at RT
- Wash with 1X PBS twice
- RNA Removal
- Add RNase Mix (20ul RNase H 10X Buffer + 2ul Riboshredder + 10ul RNase H + 168ul H2O) and incubate 1hr at 37C
- Wash with H2O twice
- Add 100nM Padlock Probe Mix with 1000X suppv2 oligos and incubate ~1hr at 60C as oven slowly decreases to 55C and held for another ~20hr
- Wrapped with parafilm to prevent evaporation
Component | Volume |
DEPC-H2O | 20.18 |
Ampligase Buffer | 10 |
Padlock Probes 320nM | 31.25 |
100nM suppv2 Oligos | 28.57 |
Ampligase | 10 |
Total | 100 |
- 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 6hr at 30C
Component | Volume |
H2O | 172 |
10X Phi29 Buffer | 20 |
25mM dNTP | 2 |
2mM aa-dUTP | 4 |
Phi 29 (low conc) | 2 |
Total | 200 |
- Wash with 10mM HEPES
- Dilute beads 1:500 in 10mM HEPES (1ul stock beads + 249ul H2O + 250ul 20mM HEPES pH 7.3)
- Make 1.2ml aliquots of stock beads and store in dcProbe box in 4C
- Sonicate for 3 minutes with microtip probe in 1.5ml tube
- Power = 3, 3sec on 2sec off
- Add to sample and incubate 5min at RT
- Wash with 10mM HEPES once
- Add 300ul BS(PEG)9 (6ul BS(PEG)9 + 294ul 20mM HEPES) 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 and store in 1X PBS at 4C overnight-->