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

Procedure

  1. Dissect flies in PBS, or collect undissected flies into 1.5 ml microcentrufuge tube.

    • 5 flies for whole body 

    • 10 flies for heads only (at least) 

  2. Prior to use, add ethanol to the RNA Wash Buffer concentrate (R1003-3-48). See bottle instructions
    Perform all steps at room temperature and centrifuge at 10,000 - 16,000 x g. 

  3. Homogenize in 300 ul RNA Lysis Buffer (R1060-1-100). 

  4. Clear lysate by centrifugation at ≥10,000 x g for 1 min. 

  5. Transfer the supernatant into a Spin-Away Filter (C1006-50-F) in a Collection Tube (C1001-50) and centrifuge at ≥10,000 x g for 1 min to remove the majority of gDNA. 

    • Save the flow-through! 

  6. Add 1 volume ethanol (95-100%) to the flow-through and mix well. 

  7. Transfer the mixture to a Zymo-Spin IIICG Column (C1006-50-G) and centrifuge 30-45 sec. 

    • Discard the flow-through. 

  8. Recommended Step: DNase I treatment (in column)

    • Add 400 ul RNA Wash Buffer to column and centrifuge for 45 sec. Discard the flow-through. 

    • Mix by inversion 5 ul DNase I and 75 ul DNA Digestion Buffer (E1010) in RNase-free tube. Add the mix directly to the column matrix. 

    • Incubate at room temperature for 15 min. Spin for 30 sec. 

  9. Add 400 ul RNA Prep Buffer to the column and centrifuge for 45 sec. Discard the flow-through.

  10. Add 700 ul RNA Wash Buffer to the column and centrifuge for 45 sec. Discard the flow-through.

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

  12. Transfer the column carefully into an RNase-free tube add DNase/RNase-Free Water directly into the column matrix and centrifuge for 45 sec.

    • Use ~10ul/whole fly of DNase/RNase-Free Water. 

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

  14. Measure the RNA concentration by NanoDrop.

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