Matt:LabNotes/2016-5-16

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Revision as of 00:33, 25 May 2016 by >Mzcai
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Test 25um Gel

Experiment Plan

  • Goal: Test whether the longer (150bp) padlock probes can diffuse through ~25-50um polyacrylamide hydrogel to generate rolonies
    • Previously worked with ~140um gel

Protocol

Day 1

  1. Prepare hydrogel mix
    1. 5% PA Gel: Standard hydrogel
  2. Attach ~25um thick adhesive to Vectabond + Bind-silane treated coverslip
  3. Add 2ul PKP2 beads diluted in 50ul H2O to coverslips right on top of 2 magnets
    • Pipette away extra H2O leaving only dried beads
  4. Inject ~50ul of 200ul hydrogel mix into each dish (underneath another coverslip)
  5. Vacuum seal bag and then pump with Argon gas
  6. Let set at RT for 30min
  7. Attach to bottom of culture dish

Day 2

  1. Wash with nf-H2O twice
  2. 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
  1. Add mix to sample and incubate for 30min at 37C
  2. Move sample to 60C and oven slowly decreases to 55C and held for ~24hr

Day 3

  1. Wash with 1X PBS once
  2. Add 1uM FISSEQ_ppRCA (1ul 200uM FISSEQ_ppRCA + 199ul 2X SSC + 30% formamide) and incubate 1hr at 55C
    • Preheated to 70C before adding
  3. Wash with 2X SSC once, 1X SSC once, and 1X PBS once
  4. 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

  1. Wash with 1X PBS once
  2. Add 200ul BS(PEG)9 (4ul BS(PEG)9 + 196ul 1X PBS) for 1hr at RT
  3. Wash with 1X PBS twice
  4. Add 1M Tris ph8.0 for 30min at RT
  5. Wash with 1X PBS twice and store in 2ml 1X PBS