RNEasy

RNEasy kdorfman Fri, 05/24/2013 - 19:30

Prepare RNA from powdered frozen tissue

Use RNeasy Plant Mini Kit (50) Qiagen 74904

  1. Add 450 µL Buffer RLT. Mix vigorously. Spin down.

  2. Transfer lysate to lilac Qiashredder in 2mL collection tube

  3. Spin 2 min, full speed

  4. Transfer flow-through (NOT PELLET) to new tube. Toss the lilac column

  5. Add 225 µL 100% EtOH to lysate. Mix by pipetting up and down

  6. Transfer entire sample to pink spin column in a 2 mL collection tube.

  7. Discard flow-through. Keep column.

  8. Add 350 µL RW1 to column. Spin 15 sec, full speed.

  9. Discard flow-through. Keep column

  10. Add 70µL RDD to 10 µL DNAse I. BE GENTLE. Put the 80 µL on the column. Incubate at room temp 15 min.

  11. Add 350 µL RW1. Spin 15 sec. full speed.

  12. Discard flow-through, reuse collection tube.

  13. Add 500 µL RPE to column. spin full speed 2 min.

  14. Put column in new capless collection tube. Discard old collection tube.

  15. Spin 1 min, full speed

  16. Add 30 µL RNAse-free water to column membrane. Spin 1 min, full speed.

  17. Repeat, with 20 µL RNAse free water.

  18. Transfer all 50 µL RNA to clean, labeled tube. Keep on ice

Reagents

Reagents kdorfman Mon, 05/27/2013 - 18:15
For RNEasy reactions
add: mL RLT
add: mL 100% EtOH
add: mL RW1
add: mL RDD
add: mL DNAse I
add: mL RPE
add: mL RNAse-free water