Daniel:Notebook/RNAFACS/2015-12-2
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PA Gel Slide Testing[edit]
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[edit]
Lysis[edit]
This protocol follows Andrew's protocols for:
For slide 1-1, do the following (in the hood)
- Remove from -80 and leave on ice block
- Add 40 uL ALS to sample; swirl to mix; incubate 10 minutes on ice block
- Add 40 uL NS; swirl to mix
- Flick off neutralized solution
RNA Scope[edit]
- Remove slide 1-2 from freezer, add 20 uL 4% paraformaldehyde in PBS to each slide; incubate 10 min at RT
- Rinse slides in 1X PBS; repeat
- Dehydration
- Incubate slides in 50% EtOH for 5 minutes at RT
- Incubate slides in 70% EtOH for 5 minutes at RT
- Incubate slides in 100% EtOH for 5 minutes at RT
- Incubate slides in 100% EtOH for 10 minutes at RT
- Rehydration
- Incubate slides in 70% EtOH for 2 minutes
- Incubate slides in 50% EtOH for 2 minutes
- Incubate slides in 1X PBS for 10 minutes
- Apply Pretreat 3
- Remove excess liquid; add 2-4 drops of pretreat 3
- Incubate for 10 min at RT (in oven)
- Flick to remove excess liquid
- Rinse in fresh 1X PBS; be sure to agitate to clean well
Multiplex Fluorescent Assay[edit]
- Prepare-Reagents
- Prepare probes by warming to 40C for 10 minutes, then cool to RT
- Briefly spin down C2 and C3 probes to collect the liquid
- Combine C2, C3, and C1 probes by pipetting into a new 1.5mL tube at 1:1:50 ratio
- Prepare 1X Wash Buffer
- Place AMP1-4 FL reagents at RT
- Ensure HybEZ OVEN and Control Tray are at 40C
- Hybridize Probe
- Flick to remove excess liquid; add 2-4 drops of probe
- Incubate for 2 hrs at 40C
- Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes
- Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes
- AMP1-FL
- Flick to remove excess liquid
- Add 2-4 drops of AMP1-FL; Incubate for 30 minutes at 40C
- Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes
- Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes
- AMP2-FL
- Flick to remove excess liquid
- Add 2-4 drops of AMP2-FL; Incubate for 15 minutes at 40C
- Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes
- Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes
- AMP3-FL
- Flick to remove excess liquid
- Add 2-4 drops of AMP3-FL; Incubate for 30 minutes at 40C
- Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes
- Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes
- AMP4-FL
- Flick to remove excess liquid
- Add 2-4 drops of AMP4-FL ALT B; Incubate for 15 minutes at 40C
- Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes
- Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes
- Counterstain and Mount
- Flick to remove excess liquid;
- Add 2-4 drops DAPI; Incubate 30 sec at RT
- Remove DAPI and immediate place 1-2 drops of fluorescent mounting medium
- Seal with a coverslip; avoid bubbles
- Store in the dark at 4C
- Recommended Viewing at 20-40X magnification
Buffers[edit]
- 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)
- 65 uL H20
- 15 uL 4M Tris-HCl
- 20 uL 2N HCl