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Total RNA Purification by Quick-RNA MiniPrep (Zymo Research)_Brian Hodge
Author: Brian Hodge, updated date: , view: 41, Q&A: 0
Tags: RNA, Fly and Drosophila

Procedure

  1. Minimum number flies to start with: 5-30 whole flies. 

    If isolating single tissue dissect flies in PBS
    Minimal number of flies:
    Heads only- 15 flies
    Guts only- 20-30 flies

  2. Homogenize in 150ul RNA lysis buffer with handheld electric homogenizer. 
    Add an additional 150ul RNA lysis buffer. 

    Centrifuge for 1 min 10000 x g

  3. First add 300ul of EtOH to the Collection Tubes
    Transfer the supernatant from step 2 into a Spin-Away filter (yellow) and place into the Collection Tube (contains EtOH). 
    Centrifuge at 10,000 x g for 1 minute to remove the majority of genomic DNA. 

  4. Transfer the mixture to a Zymo-Spin IIICG Column in a Collection Tube and centrifuge for 45 seconds (10,000 x g)

  5. Discard the flow-through

  6. Add 400 ul RNA Wash Buffer (Make sure EtOH has been added) to the column and centrifuge for 45 seconds (10,000 x g) 

  7. Discard the flow-through

  8. Mix DNase I Reaction Buffer. 80ul buffer needed per sample (10 samples, 800 ul buffer) 
    Add 5ul DNase I and 75ul DNA Digestion Buffer in a fresh tube. Mix. 
    Add 80ul of the DNase I Reaction Buffer to the coumn matrix and incubate at room temperature for at least 15 minutes. (Can go longer) 

  9. Add 400ul RNA Prep Buffer to the column and centrifuge. 

  10. Discard the flow-through

  11. Add 700 ul RNA Wash Buffer to the column and centrifuge for 45 seconds. 

  12. Discard the flow-through. 

  13. Add 400 ul RNA Wash Buffer and centrifuge the column for 2 minutes to ensure complete removal of the wash buffer. 

  14. Place the column into an RNase-free tube. Add 30ul DNase/RNase-Free water directly to the column matrix.
    Centrifuge at 10,000 x g for 45 seconds.

  15. Eluted RNA can be used immediately or stored at -80C

    Measure the RNA concentration by NanoDrop:
    1ul of sample
    Set for "RNA-40"
    "Blank" button
    sample= "Measure" 


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