Matt:LabNotes/2016-4-28: Difference between revisions
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>Mzcai m (→Protocol) |
>Mzcai m (→Day 2) |
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===Day 3=== | ===Day 3=== | ||
#Wash with 1X PBS | #Wash with 1X PBS once | ||
#Add 200ul BS(PEG)9 (4ul BS(PEG)9 + 196ul 1X PBS) for 1hr at RT | #Add 200ul BS(PEG)9 (4ul BS(PEG)9 + 196ul 1X PBS) for 1hr at RT | ||
#Wash with 1X PBS twice | #Wash with 1X PBS twice | ||
#Add 1M Tris ph8.0 for 30min at RT | #Add 1M Tris ph8.0 for 30min at RT | ||
#Wash with 1X PBS twice and store in 2ml 1X PBS | |||
==Imaging== | ==Imaging== | ||
*Used Gain and Laser power usually used for DARTFISH decoding | |||
**ch00: Cy5 | |||
**ch01: Cy3 | |||
**ch02: BF | |||
*Pos1: NegCtrl with no beads | |||
*Pos2: Scattered beads | |||
*Pos3: Scattered beads | |||
====Before Hybridization==== | |||
*No fluorescent signal anywhere | |||
====Hybridize FISGA_Adpt Cy3==== | |||
#Preheat 200ul 0.5uM FISGA_Adpt_Cy3 in 30% formamide 2X SSC to 75C | |||
#Add 200ul to each dish and let sit for 10min at RT | |||
#Wash twice with 2X SSC | |||
====Strip==== | |||
#Preheat 80% formamide 2X SSC to 75C | |||
#Add 200ul to Dish1 (PosCtrl) and let sit for 15min at RT | |||
#Wash twice with 1X PBS | |||
#Add 500ul 2x SSC to image | |||
====Hybridize Cycle5 Cy3 & Cy5==== | |||
*Only Cy5 should show signal | |||
#Preheat 200ul 0.5uM Cycle04 in 30% formamide 2X SSC to 75C | |||
#Add 200ul to each dish and let sit for 10min at RT | |||
#Wash twice with 2X SSC |
Revision as of 05:26, 4 May 2016
Test Diffusion of 5% PA 120um Gel Using CA12kNov2014_V4 Probes
- Using PKP2 Beads as targets
Experiment Plan
- Goal: Test whether the longer (150bp) padlock probes can diffuse through ~120um polyacrylamide hydrogel to generate rolonies
- Previously PKP2_controlPP worked but in that experiment:
- The concentration was 33X higher than in actual DARTFISH probeset (1nM vs 30pM)
- PKP2_controlPP is half the length (~70bp vs 150bp)
- Previously PKP2_controlPP worked but in that experiment:
Protocol
Day 1
- Prepare hydrogel mix
- 5% PA Gel: Standard hydrogel
- Attach ~120um thick adhesive to Vectabond + Bind-silane treated coverslip
- Add 6ul PKP2 beads diluted in 50ul H2O to coverslips right on top of 2 magnets
- Pipette away extra H2O leaving only dried beads
- Inject ~50ul of 200ul hydrogel mix into each dish (underneath another coverslip)
- Vacuum seal bag and then pump with Argon gas
- Let set at RT for 30min
- Attach to bottom of culture dish
- Wash with nf-H2O twice
- Prepare Ampligase mix on ice
- Preheat H2O + Padlock probes to 85C and then snap cool before adding Ampligase buffer and enzyme
Component | Volume |
DEPC-H2O | 52 |
Ampligase Buffer | 10 |
Batch 15 368nM | 28 |
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 2
- 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 15hr at 30C
Component | Volume |
H2O | 174 |
10X Phi29 Buffer | 20 |
25mM dNTP | 2 |
4mM aa-dUTP | 2 |
Phi 29 (low conc) | 2 |
Total | 200 |
Day 3
- Wash with 1X PBS once
- Add 200ul BS(PEG)9 (4ul BS(PEG)9 + 196ul 1X PBS) for 1hr at RT
- Wash with 1X PBS twice
- Add 1M Tris ph8.0 for 30min at RT
- Wash with 1X PBS twice and store in 2ml 1X PBS
Imaging
- Used Gain and Laser power usually used for DARTFISH decoding
- ch00: Cy5
- ch01: Cy3
- ch02: BF
- Pos1: NegCtrl with no beads
- Pos2: Scattered beads
- Pos3: Scattered beads
Before Hybridization
- No fluorescent signal anywhere
Hybridize FISGA_Adpt Cy3
- Preheat 200ul 0.5uM FISGA_Adpt_Cy3 in 30% formamide 2X SSC to 75C
- Add 200ul to each dish and let sit for 10min at RT
- Wash twice with 2X SSC
Strip
- Preheat 80% formamide 2X SSC to 75C
- Add 200ul to Dish1 (PosCtrl) and let sit for 15min at RT
- Wash twice with 1X PBS
- Add 500ul 2x SSC to image
Hybridize Cycle5 Cy3 & Cy5
- Only Cy5 should show signal
- Preheat 200ul 0.5uM Cycle04 in 30% formamide 2X SSC to 75C
- Add 200ul to each dish and let sit for 10min at RT
- Wash twice with 2X SSC