NEB DNAse I Reaction Protocol

NEB DNAse I A Typical DNase I Reaction Protocol (M0303) | NEB

1. Clean the UV-cabinet surfaces with 70% ethanol and then with RNAzap to remove RNAs.
2. Set up the following reaction on ice in 1.5 mL Eppendorf (autoclaved):

Component	Amount
RNA	20 pmol
DNase I Reaction Buffer (10X)	10 uL
DNAse I (RNase-free)	1 uL (2 units)
Nuclease-free H2O	to 100 uL
Original protocol calls for 10 ug RNA. Assuming its size of 1000-2000 nt, it amounts to 20 pmol.
3. Incubate at 37°C for 10 minutes.
4. If not using it further (otherwise proceed to purification):
- Add 1 µl of 0.5 M EDTA (to a final concentration of 5 mM).
- Heat inactivate at 75°C for 10 minutes.

GeneJet Kit: Genomic DNA Removal from RNA Preparations

GeneJet RNA Purification Kit Optimized for RNAs > 200 nt

1. Adjust the volume of the reaction mixture to 100 µL with Water, nuclease-free (included)
2. Add 300 µL of Lysis Buffer without β-mercaptoethanol or DTT. Mix thoroughly by vortexing or pipetting.
3. Add 180 µL of ethanol (96-100%) and mix by pipetting.
4. Transfer the mixture to the GeneJET RNA Purification Column inserted in a collection tube. Centrifuge the column for 1 min at ≥12000 × g. Discard the collection tube containing the flow-through solution.
5. Place the GeneJET RNA Purification Column into a new 2 mL collection tube (included).
   • Note. Close the bag with GeneJET RNA Purification Columns tightly after each use!
6. Add 700 µL of Wash Buffer 1 to the GeneJET RNA Purification Column and centrifuge for 1 min at ≥12000 × g. Discard the flowthrough and place the purification column back into the collection tube.
7. Add 600 µL of Wash Buffer 2 to the GeneJET RNA Purification Column and centrifuge for 1 min at ≥12000 × g. Discard the flowthrough and place the purification column back into the collection tube.
8. Add 250 µL of Wash Buffer 2 to the GeneJET RNA Purification Column and centrifuge for 3 min at ≥12000 × g. Discard the collection tube containing the flow-through solution and transfer the GeneJET RNA Purification Column to a sterile 1.5 mL RNase-free microcentrifuge tube (included).
9. Add 50 µL of Water, nuclease-free to the center of the GeneJET RNA Purification Column membrane. Centrifuge for 1 min at ≥12000 × g to elute RNA.
10. Repeat Step 13.
11. Discard the purification column. Use the purified RNA for downstream applications or store RNA at -20°C or -70°C until use.

Direct-zol RNA Miniprep protocol

Zymo Research Direct-zol™ RNA Miniprep Perform all steps at room temperature and centrifuge at 10,000 - 15,000 g. Consider increasing centrifugation duration if sample purity is low.

1. Adjust the volume of transcription product to 100 uL with nuclease-free water.
2. Add 300 uL TRI Reagent and vortex.
   • These samples can be stored frozen for later processing.
3. Add 400 uL 100% ethanol.
4. Transfer the mixture into a Zymo-Spin™ IICR Column in a Collection Tube and centrifuge for 1 min.
5. Transfer the column into a new collection tube.
   • To process samples > 700 μl, reload the column and repeat this step.
   • Make sure the column does not touch the original collection tube during the transfer to prevent buffer carryover.
6. (Optional) DNAse I treatment:
   1. Add 400 μl RNA Wash Buffer to the column and centrifuge.
   2. In an RNase-free tube, add 5 μl DNase I and 75 μl DNA Digestion Buffer, and mix by gentle inversion. Add the mix directly to the column matrix.
   3. Incubate at room temperature (20-30°C) for 15 min.
7. Repeat twice:
   1. Add 400 μl Direct-zol™ RNA PreWash to the column and **centrifuge for 1 min.
   2. Discard the flow-through.
8. Add 700 μl RNA Wash Buffer to the column and centrifuge for 2 min to ensure complete removal of the wash buffer.
9. Transfer the column carefully into an RNase-free tube (not included).
10. To elute RNA, add 50 μl of nuclease-free water directly to the column matrix and **centrifuge for 1 min.
    • Alternatively, for highly concentrated RNA use ≥ 25 μl elution.
    • The eluted RNA can be used immediately or stored frozen.