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Update mRNA stability protocol to use reverse transfection method
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@ -42,8 +42,9 @@ transfection:
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reagent: "Lipofectamine RNAiMAX"
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solution_1: "25 µL Opti-MEM + 1 µL Lipofectamine RNAiMAX per well"
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solution_2: "25 µL Opti-MEM + 1 µL of 10 µM siRNA per well"
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final_volume_per_well: "50 µL transfection mix + 0.9 mL media"
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final_volume_per_well: "50 µL transfection mix + 750 µL media + 200 µL cell suspension = 1 mL total"
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incubation_time: "5 min"
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method: "Reverse transfection (siRNA complexes added before cells)"
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siRNA:
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- name: "siYBX1"
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concentration: "10 µM stock, 10 nM final"
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@ -75,14 +76,8 @@ instruments:
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# 1️⃣ Experiment Timeline & Execution
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## Day 1: 2025-05-07
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- [ ] Seed cells in 24-well plates (one plate per cell line):
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- Huh7: 5 × 10⁴ cells / well
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- HepG2: 6 × 10⁴ cells / well
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- [ ] Prepare plates and label wells according to condition map
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- [ ] Incubate O/N at 37°C + 5% CO₂
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## Day 2: 2025-05-08
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## Day 1: 2025-05-07 (Reverse Transfection)
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- [ ] Prepare 24-well plates and label according to condition map (one plate per cell line)
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- [ ] Prepare transfection master mixes for both plates (48 wells + 4 extra = 52 wells total):
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**Solution 1 (common for all wells in both plates):**
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@ -105,22 +100,27 @@ instruments:
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- For siCTRL wells: Mix 650 µL of Solution 1 with 650 µL of Solution 2B
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- Incubate combined solutions for 5 min at RT
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- [ ] Prepare cells for transfection:
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- Aspirate spent medium from wells
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- Add 0.9 mL fresh complete medium to each well (use appropriate media for each cell line)
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- [ ] Add 50 µL of transfection complexes to each appropriate well of empty 24-well plates
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- [ ] Add transfection complexes:
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- Add 50 µL transfection complex to each appropriate well
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- Gently rock plate to distribute complexes
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- [ ] Prepare cell suspensions from confluent flasks:
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- Wash confluent flasks with 10 mL PBS
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- Add 1 mL trypsin to each flask and incubate ~10 min until cells detach
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- Quench with 9 mL complete media (use correct media for each cell line)
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- Resulting cell suspension: approximately 10 mL from each confluent flask
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- [ ] Perform reverse transfection:
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- Add 750 µL appropriate complete media to each well containing transfection complexes
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- Add 200 µL of cell suspension from confluent flask to each well (total 1 mL per well)
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- Gently rock plates to distribute cells and complexes
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- Return plates to 37°C + 5% CO₂ incubator
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## Day 3: 2025-05-09
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## Day 3: 2025-05-09 (48h post-transfection)
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- [ ] Collect one well from each condition for knockdown verification:
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- Extract RNA using TRIzol
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- RT-qPCR for YBX1 (vs control wells)
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- Confirm >70% knockdown
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## Day 4: 2025-05-10
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## Day 4: 2025-05-10 (72h post-transfection)
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- [ ] Preparation for actinomycin D treatment:
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- Label tubes for all timepoints
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- Thaw actinomycin D (protect from light)
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