Trizol and PureLink RNA Mini Kit RNA purification protocol

Following the collection of the cells, perform the following steps to isolate the sample. 

1. Incubate the lysate with TRIzol Reagent at room temperature for 5 minutes to allow complete dissociation of nucleoprotein complexes. 

2. Add 0.2 mL chloroform per 1 mL TRIzol Reagent used. Shake the tube vigorously by hand for 15 seconds. 

Note: Vortexing may increase DNA contamination of your RNA sample. Avoid vortexing if your downstream application is sensitive to the presence of DNA or perform an on-column DNase-digestion step during RNA purification (page 63) or after purification (page 65). 

3. Incubate at room temperature for 2–3 minutes. 

4. Centrifuge the sample at 12,000 × for 15 minutes at 4oC. 

Note: After centrifugation, the mixture separates into a lower, red phenol-chloroform phase, an interphase, and a colorless upper aqueous phase which contains the RNA. The volume of the aqueous upper phase is ~600 μL. 

5. Transfer ~400 μL of the colorless, upper phase containing the RNA to a fresh RNase–free tube. 

6. Add an equal volume 70% ethanol to obtain a final ethanol concentration of 35%. Vortex to mix well. 

7. Invert the tube to disperse any visible precipitate that may form after adding ethanol. 

8. Transfer ≤700 μL of sample (see previous page) to a Spin Cartridge (with a Collection Tube). 

9. Centrifuge at 12,000 × for 15 seconds at room temperature. Discard the flow-through and reinsert the Spin Cartridge into the same Collection Tube. 

10. Repeat Steps 1–2 until the entire sample is processed. 

11. Add 350 μL Wash Buffer I to the Spin Cartridge containing the bound RNA (see sample–specific protocol). Centrifuge at 12,000 × g for 15 seconds at room temperature. Discard the flow-through and the Collection Tube. Insert the Spin Cartridge into a new Collection Tube. 

12. Add 80 μL PureLinkDNase mixture directly onto the surface of the Spin Cartridge membrane. Prepare the mixture as follows (this recipe is enough to treat 1 column, scale up accordingly):

10X Reaction Buffer                    8ul

Resuspended DNAse                 10ul

RNAse free water                     62ul

13. Incubate at room temperature for 15 minutes. 

14. Add 350 μL Wash Buffer I to the Spin Cartridge. Centrifuge at 12,000 x g for 15 seconds at room temperature. Discard flow-through and the Collection Tube and insert the Spin Cartridge into a new Collection Tube. 

15. Add 500 μL Wash Buffer II with ethanol (page 11) to the Spin Cartridge. 

16. Centrifuge at 12,000 x g for 15 seconds at room temperature. Discard flow-through and reinsert the Spin Cartridge into the same Collection Tube. 

17. Repeat Steps 5–6, once

18. Centrifuge the Spin Cartridge at 12,000 × g for 1 minute to dry the membrane with bound RNA. Discard Collection Tube and insert the Spin Cartridge into a Recovery Tube. 

19. Add 30 μL–100 μL RNase–Free Water to the center of the Spin Cartridge. 

20. Incubate at room temperature for 1 minute. 

21. Centrifuge Spin Cartridge and Recovery Tube for 1 minute at ≥12,000 x g at room temperature. 

22. Store your purified RNA (see page 4), or proceed to Analyzing RNA Yield and Quality.