Daniel:Notebook/RNAFACS/2015-12-1

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Revision as of 19:42, 1 December 2015 by >Djacobse (→‎Multiplex Fluorescent Assay)
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PA Gel Slide Testing

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Time to test these gels on the slides. I'm going to test the protocol with and without nuclear lysis.

Samples Matrix I'll be using samples 1-1 and 1-2.

Protocol

Lysis

This protocol follows Andrew's protocols for:

For slide 1-1, do the following (in the hood)

  1. Remove from -80 and leave on ice block
  2. Add 10 uL ALS to sample; swirl to mix; incubate 10 minutes on ice block
  3. Add 10 uL NS; swirl to mix
  4. Flick off neutralized solution

RNA Scope

  1. Remove slide 1-2 from freezer, add 20 uL 4% paraformaldehyde in PBS to each slide
  2. Rinse slides in 1X PBS; repeat
  3. Dehydration
    1. Incubate slides in 50% EtOH for 5 minutes at RT
    2. Incubate slides in 70% EtOH for 5 minutes at RT
    3. Incubate slides in 100% EtOH for 5 minutes at RT
    4. Incubate slides in 100% EtOH for 10 minutes at RT
  4. Rehydration
    1. Incubate slides in 70% EtOH for 2 minutes
    2. Incubate slides in 50% EtOH for 2 minutes
    3. Incubate slides in 1X PBS for 10 minutes
  5. Apply Pretreat 3
    1. Remove excess liquid; add 2-4 drops of pretreat 3
    2. Incubate for 10 min at RT (in oven)
    3. Flick to remove excess liquid
    4. Rinse in fresh 1X PBS; be sure to agitate to clean well

Multiplex Fluorescent Assay

  1. Prepare-Reagents
    1. Prepare probes by warming to 40C for 10 minutes, then cool to RT
    2. Briefly spin down C2 and C3 probes to collect the liquid
    3. Combine C2, C3, and C1 probes by pipetting into a new 1.5mL tube at 1:1:50 ratio
    4. Prepare 1X Wash Buffer
    5. Place AMP1-4 FL reagents at RT
    6. Ensure HybEZ OVEN and Control Tray are at 40C
  2. Hybridize Probe
    1. Flick to remove excess liquid; add 2-4 drops of probe
    2. Incubate for 2 hrs at 40C
    3. Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes
    4. Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes
  3. AMP1-FL
    1. Flick to remove excess liquid
    2. Add 2-4 drops of AMP1-FL; Incubate for 30 minutes at 40C
    3. Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes
    4. Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes
  4. AMP2-FL
    1. Flick to remove excess liquid
    2. Add 2-4 drops of AMP2-FL; Incubate for 15 minutes at 40C
    3. Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes
    4. Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes
  5. AMP3-FL
    1. Flick to remove excess liquid
    2. Add 2-4 drops of AMP3-FL; Incubate for 30 minutes at 40C
    3. Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes
    4. Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes
  6. AMP4-FL
    1. Flick to remove excess liquid
    2. Add 2-4 drops of AMP4-FL ALT B; Incubate for 15 minutes at 40C
    3. Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes
    4. Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes
  7. Counterstain and Mount
    1. Flick to remove excess liquid;
    2. Add 2-4 drops DAPI; Incubate 30 sec at RT
    3. Remove DAPI and immediate place 1-2 drops of fluorescent mounting medium
    4. Seal with a coverslip; avoid bubbles
  8. Store in the dark at 4C
  9. Recommended Viewing at 20-40X magnification

Buffers

  • Alkaline Lysis Solution (ALS)
    • 80 ul Nuclease free H2O.
    • 8 ul 5 M KOH.
    • 10 ul 1 M DTT.
    • 2 ul 0.5 M EDTA
  • Neutralization Solution (NS)
    • 3 mL H20
    • 3 mL 2M Tris-HCl
    • 4 mL 1N HCl