Matt:LabNotes/2015-8-4: Difference between revisions
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>Mzcai |
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| Ampligase Buffer||20 | | Ampligase Buffer||20 | ||
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| [[ | | [[Matt:JustinLabNotes/2015-7-9#Qubit_Quantification | Padlock Probes 337nM]]||39 | ||
|- | |- | ||
| Ampligase||20 | | Ampligase||20 | ||
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#*Preheated to 65C before adding | #*Preheated to 65C 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 6hr at 30C | ||
{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''Component''' | | align="center" style="background:#f0f0f0;"|'''Component''' | ||
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| Total||200 | | 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 | |||
===Results=== | |||
====Pos1: Gain 550==== | |||
*Fluorescent beads saturated intensity | |||
*All Rolonies | |||
[[File:MAX_20150804_BA8DARTFISH_FidBeads_Gain550_Pos1_ch00.jpg|650px]] | |||
*Fidicual Beads | |||
[[File:MAX_20150804_BA8DARTFISH_FidBeads_Gain550_Pos1_ch01.jpg|650px]] | |||
====Pos2: Gain 400==== | |||
*Decreased gain of 488 channel | |||
*All Rolonies | |||
[[File:MAX_20150804_BA8DARTFISH_FidBeads_Gain400_Pos2_ch00.jpg|650px]] | |||
*Fidicual Beads | |||
[[File:MAX_20150804_BA8DARTFISH_FidBeads_Gain400_Pos2_ch01.jpg|650px]] |
Latest revision as of 02:42, 14 August 2015
BA8 Section DARTFISH with Fiducial Beads[edit]
- 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[edit]
- 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 |
Padlock Probes 337nM | 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 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
Results[edit]
Pos1: Gain 550[edit]
- Fluorescent beads saturated intensity
- All Rolonies
File:MAX 20150804 BA8DARTFISH FidBeads Gain550 Pos1 ch00.jpg
- Fidicual Beads
File:MAX 20150804 BA8DARTFISH FidBeads Gain550 Pos1 ch01.jpg
Pos2: Gain 400[edit]
- Decreased gain of 488 channel
- All Rolonies
File:MAX 20150804 BA8DARTFISH FidBeads Gain400 Pos2 ch00.jpg
- Fidicual Beads
File:MAX 20150804 BA8DARTFISH FidBeads Gain400 Pos2 ch01.jpg