Matt:LabNotes/2016-5-16: Difference between revisions
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>Mzcai (Created page with "=Test 140um Gel= *Like this test but thinner gel *Using PKP2 Beads as targets ==Experiment Plan== *Goal: Test whether ...") |
>Mzcai m (→Day 2) |
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#Add mix to sample and incubate for 30min at 37C | #Add mix to sample and incubate for 30min at 37C | ||
#Move sample to 60C and oven slowly decreases to 55C and held for ~24hr | #Move sample to 60C and oven slowly decreases to 55C and held for ~24hr | ||
===Day | ===Day 3=== | ||
#Wash with 1X PBS once | #Wash with 1X PBS once | ||
#Add 1uM FISSEQ_ppRCA (1ul 200uM FISSEQ_ppRCA + 199ul 2X SSC + 30% formamide) and incubate 1hr at 55C | #Add 1uM FISSEQ_ppRCA (1ul 200uM FISSEQ_ppRCA + 199ul 2X SSC + 30% formamide) and incubate 1hr at 55C | ||
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| Total||200 | | Total||200 | ||
|} | |} | ||
===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) for 1hr at RT | #Add 200ul BS(PEG)9 (4ul BS(PEG)9 + 196ul 1X PBS) for 1hr at RT | ||
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#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 | #Wash with 1X PBS twice and store in 2ml 1X PBS | ||
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==Imaging== | ==Imaging== | ||
*Used Gain and Laser power usually used for DARTFISH decoding | *Used Gain and Laser power usually used for DARTFISH decoding |
Revision as of 22:43, 18 May 2016
Test 140um Gel
- Like this test but thinner gel
- 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 ~25um thick adhesive to Vectabond + Bind-silane treated coverslip
- Add 2ul 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
Day 2
- 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 | 37 |
Ampligase Buffer | 10 |
Batch 14 237nM | 43 |
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 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 4
- 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