Matt:LabNotes/2015-8-4: Difference between revisions
Jump to navigation
Jump to search
>Mzcai |
>Mzcai |
||
Line 54: | Line 54: | ||
#*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 H2O twice | ||
#Add Ampligase Mix (no suppressor) and incubate ~3hr at 60C as oven slowly decreases to 55C and held for another ~16hr | #Add 66nM Ampligase Mix (no suppressor) and incubate ~3hr at 60C as oven slowly decreases to 55C and held for another ~16hr | ||
#*Wrapped with parafilm to prevent evaporation | #*Wrapped with parafilm to prevent evaporation (works very well) | ||
{| {{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||121 | ||
|- | |- | ||
| Ampligase Buffer||20 | | Ampligase Buffer||20 | ||
|- | |- | ||
| Padlock Probes|| | | [[Padlock Probes 337nM | Matt:JustinLabNotes/2015-7-9#Qubit_Quantification]]||39 | ||
|- | |- | ||
| Ampligase||20 | | Ampligase||20 | ||
|- | |||
| Total||200 | |||
|} | |||
#Wash with 1X PBS once | |||
#Add 1uM FISSEQ_ppRCA (0.5ul 200uM FISSEQ_ppRCA + 199.5ul 2X SSC + 30% formamide) and incubate 1hr at 55C | |||
#*Preheated to 65C before adding | |||
#Wash with 2X SSC once, 1X SSC once, and 1X PBS once | |||
#Add RCA mix and incubate __hr at 30C | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Component''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume''' | |||
|- | |||
| H2O||172 | |||
|- | |||
| 10X Phi29 Buffer||20 | |||
|- | |||
| 25mM dNTP||2 | |||
|- | |||
| 2mM aa-dUTP||4 | |||
|- | |||
| Phi 29 (low conc)||2 | |||
|- | |- | ||
| Total||200 | | Total||200 | ||
|} | |} |
Revision as of 20:18, 6 August 2015
BA8 Section DARTFISH with Fiducial Beads
- Try again: Matt:LabNotes/2015-8-3
- with fresh PFA
- Practice DARTFISH on BA8 tissue sections
- Use FluoSpheres Amine-Modified Microspheres 0.2um (505/515) as fiducial markers
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
- 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
- Still had leak so used gap-filling adhesive 200 to seal
- Add 0.25% TX-100 in 2XSSPE and incubate 10min at RT
- 0.25% TX-100 in 2XSSPE tube had crack in cap so not completely sealed
- Wash with nuclease-free H2O three times
- Add 0.01% Pepsin in 0.1N HCl and incubate for 5min at 37C
- 2ul 1%Pepsin + 10ul 2N HCl + 188ul H2O
- Wash with nuclease-free PBS three times
- Can already see tissue is degrading
- 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 ~15hr at 37C
- Tissue is slightly degraded but mostly okay
- 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 66nM Ampligase Mix (no suppressor) and incubate ~3hr at 60C as oven slowly decreases to 55C and held for another ~16hr
- Wrapped with parafilm to prevent evaporation (works very well)
Component | Volume |
DEPC-H2O | 121 |
Ampligase Buffer | 20 |
Matt:JustinLabNotes/2015-7-9#Qubit_Quantification | 39 |
Ampligase | 20 |
Total | 200 |
- Wash with 1X PBS once
- Add 1uM FISSEQ_ppRCA (0.5ul 200uM FISSEQ_ppRCA + 199.5ul 2X SSC + 30% formamide) and incubate 1hr at 55C
- Preheated to 65C before adding
- Wash with 2X SSC once, 1X SSC once, and 1X PBS once
- Add RCA mix and incubate __hr at 30C
Component | Volume |
H2O | 172 |
10X Phi29 Buffer | 20 |
25mM dNTP | 2 |
2mM aa-dUTP | 4 |
Phi 29 (low conc) | 2 |
Total | 200 |