6/6/2019 RNA Extraction Protocol - Google Docs

Joanna Ladopoulou

RNA Extraction Protocol

Prep Steps

1. Wipe down surfaces, pipettes & tips, tube stands.

2. Make sure you have your lab notebook with your table inside.

3. Prep hood. Make waste containers and line up pessels.

4. Add BME to RLT/C buffer. For 8 samples: 4000uL buffer RLT and 40uL BME.

5. Prepare a tube with RNAse-free ethanol

6. Thaw DNAse I in a bucket of ice

7. Label Tubes: 8 clear, supplied, fully labeled. The rest number 1-8. Order:

a. Purple,

b. not supplied tubes,

c. pink,

d. collection tubes,

e. final labelled ones.

8. Get liquid nitrogen. Put in it the tubes you want to extract RNA from.

https://docs.google.com/document/d/1hBtcf44O3crBcaEy2KmS-la0QIsBrae2T-pUMBczBJU/edit# 1/2
6/6/2019 RNA Extraction Protocol - Google Docs

Joanna Ladopoulou

Protocol

1. Take tube from liquid nitrogen bucket. Tap down. Keep dipping into liquid N. Open the lid.

2. Freeze the pessel. Give it a 2 second grind. Keep in liquid N.

3. Fill up pipette with 450uL buffer RLT/C (650 if there’s a lot of tissue), take out the tube, and
put in the buffer. Leave aside. Do the same to another tube.

4. Grind up well, close lid, and put conte pessel in discarded pile. Repeat for the other one.
Repeat steps 1-3 for all 8 samples.

5. Incubate for 5 mins with tubes laid flat.

6. Transfer to the purple column and centrifuge for 2 min at full speed.

7. Careful with the pellet! Transfer flow through to quick labelled tube. Discard purple tubes.

8. Add 0.5 volume (225uL) ethanol. Pipette mix.

9. Transfer (usually 650uL) to pink tube. Close lid gently. Centrifuge 15s, 10,000 rpm.

10. Discard flow through and put column back into collection tube.

11. DNAse treatment:

a. Add 350uL buffer RW1 to pink column. Gently close lid. Centrifuge 15s, 10,00rpm.
Discard flow through.

b. Insert 560uL buffer RDD into DNAse tube. Gently invert to mix.

c. Add 80uL DNAse mix directly onto the membrane of the pink columns!

d. Rest for 15min.

e. Add 350uL buffer RW1 to column. Centrifuge 15s, 10,000rpm. Discard flow through.

12. Add 500uL buffer RPE. Centrifuge 15s, 10,000rpm. Discard flow through.

13. Add 500uL buffer RPE. Centrifuge 2min, 10,000rpm. Discard flow through.

14. Place in empty collection tube. Centrifuge 1min, 10,000rpm.

15. Place pink column in fully labeled tube. Add 30uL DEPC to membrane.

16. Incubate for 5 min.

17. Centrifuge 1min, 10,000rpm. Place on ice immediately.

https://docs.google.com/document/d/1hBtcf44O3crBcaEy2KmS-la0QIsBrae2T-pUMBczBJU/edit# 2/2