Aug 08, 2025
RNAi synthesis for soaking
- Casper Zhao1
- 1westlake university
Protocol Citation: Casper Zhao 2025. RNAi synthesis for soaking. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6z985gqe/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 08, 2025
Last Modified: August 08, 2025
Protocol Integer ID: 224300
Keywords: rnai synthesis for elegan, soaking rnai synthesis, rnai synthesis, elegan
Abstract
RNAi synthesis for elegans soaking
Materials
Primers (synthesized by company), cDNA for PCR, ddH₂O, 2×Phanta Max Master Mix (Vazyme), T7 RNAi Transcription Kit (Vazyme), NTP Mix, 10× Transcription Buffer, T7 Enzyme Mix, DNase I, RNase-free H₂O, RNA Clean Beads (for magnetic bead purification), 80% ethanol (prepared with RNase-free H₂O), isopropanol (if transcript <100 bp), PCR tubes, magnetic stand, pipettes.
Troubleshooting
Safety warnings
Be careful not to stir the beads when sucking the supernatant.
Do not suck the beads.
Dry until there is no gleaming water on the surface of the magnetic beads, excessive drying will affect the elution of RNA.
In order to avoid the influence of magnetic beads on subsequent experiments, when transferring the product, please reserve 1 - 2 μl solution to prevent absorption of the magnetic beads.
Design primers
Copy CDS sequences of targeted genes.
* If there are multiple transcripts, pick the shortest one that could apply to all the transcripts.
Design primers.
* 58–60 °C, ~20 bp
- If the CDS is shorter than 2000 bp, design primers to cover ~80% of the CDS; if the CDS is longer than 2000 bp, design primers to cover ~2000 bp.
Add T7 sequence in the 5' prime of both forward and reverse primers.
Primers synthesized by company.
DNA template synthesis
Set up experiment with necessary material: primers, template (cDNA for PCR, ddH₂O, enzyme (2×Phanta Max Master Mix, Vazyme).
Prepare reaction system using following sheet.
2×Phanta Max Master Mix 12.5 μL
Primer F 1 μL
Primer R 1 μL
Template 0.5 μL
ddH₂O up to 25 μL
PCR program (list as follow) for replicating target DNA.
95°C 3 min
(95°C 15 sec
60°C 15 sec
72°C 1 min) 35 cycles
72°C 5 min
4°C Forever
Electrophoresis of 10 μL product to check correctness.
RNA transcription and dsRNA purification
Prepare reaction system using following sheet.
NTP Mix 8 μL
10× Transcription Buffer 2 μL
T7 Enzyme Mix 2 μL
DNA template 2 μL
RNase-free H₂O 6 μL
PCR program for transcription: 37°C, 4h or overnight.
Annealing to form dsRNA: 72°C, 10 min; let naturally cool to room temperature, ~10 min.
Digest DNA template.
Transcription Product 20 μL
DNase I 1 μL
RNase-free H₂O 19 μL
37°C, 30 min.
dsRNA purification following instruction.
The RNA product can be purified by the methods of magnetic beads, column, phenol/chloroform extraction or gel recovery. This kit is recommended to purify RNA by magnetic beads.
Magnetic beads purification can quickly and efficiently remove proteins, free nucleic acids and salts. The 80% ethanol used for purification needs to be prepared with RNase-free H₂O (self-prepared).
Take the RNA Clean Beads out of 4°C and equilibrate for 30 min at room temperature. Please invert or vortex before use.
Add 80 μl magnetic beads solution to the transcript and pipette 10 times or more to mix the solution thoroughly.
▲ If fragment length of the transcript is less than 100 bp, add 200 μl isopropanol and mix thoroughly.
Incubate for 8 min at room temperature to allow RNA to bind to the beads.
Place the PCR tube on the magnetic stand for about 5 min, and carefully remove the supernatant after the solution is clarified. Please be careful not to stir the beads when sucking the supernatant.
Keep the PCR tube on the magnetic stand and add 200 μl 80% ethanol prepared freshly. Be careful not to stir the beads, incubate for 30 sec at room temperature, and carefully remove the supernatant. Repeat this step once.
Open the lip and dry the magnetic beads with air for 5 - 10 min.
▲ Dry until there is no gleaming water on the surface of the magnetic beads, excessive drying will affect the elution of RNA.
Remove the PCR tube from the magnetic stand, add 40 μl RNase-free H₂O, use pipette to rinse the magnetic beads on the tube wall, mix thoroughly, and incubate for 3 min at room temperature.
Place the PCR tube on the magnetic stand, and carefully remove the supernatant to a new RNase-free EP tube after the solution is clarified. Do not suck the beads.
▲ In order to avoid the influence of magnetic beads on subsequent experiments, when transferring the product, please reserve 1 - 2 μl solution to prevent absorption of the magnetic beads.
Measure the absorbance of the product at A₂₆₀, determine its concentration, and store the purified product at -20°C.
Detect the transcripts by electrophoresis: add 1 μL of product to 9 μL ddH₂O and run gel to check correctness.