Improve Research Reproducibility A Bio-protocol resource
tooltip

RNA interference

SC Shawn C. Chafe
SD Shoukat Dedhar

Published: Feb 24, 2022 Views: 644

Original research article

The authors used this protocol in:

https://doi.org/10.1126/sciadv.abj0364

Aug 26, 2021

pdf PDF PDF
ask Ask a question
How to cite
Favorite
Cited by

siRNA Transfection with siLentFect (6-well)


Required reagents:

  • siLentfect (Bio-Rad #1703360)
  • siRNA to gene of interest (20 mM – Qiagen)
  • OPTI-MEM (Thermofisher Scientific #31985070)
  • 6-well plates (VWR # CA62406-161)

 

Protocol:

  1. siRNA should be 20 μM
  2. Label tubes and plates. Let reagents and OPTI get to RT before use. Vortex reagents.
  3. Add 7.5 μL siRNA and 500 μL Opti-MEM. Vortex.
  4. To another tube: add 2.5 μL siLentfect and 500 μL Opti. Vortex.
  5. Combine 3 and 4 for 20 minutes.
  6. Meanwhile, split cells so that they will be ~75% confluent at plating (Cell type dependent and requires titration to determine this value. 5x104 cells/cm2 is a good starting point).
  7. Add 1 mL of cells at desired concentration (see #6) to each well of 6-well. Add the combined siRNA mix and pipette up and down.
  8. Change media or split the next day. 
  9. Assay efficiency of gene depletion 72 hours post-transfection.
Copyright: Content may be subjected to copyright.

Do you have any questions about this protocol?

Post your question to gather feedback from the community. We will also invite the authors of this article to respond.

0/150

tip Tips for asking effective questions

+ Description

Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images.

post Post a Question
0 Q&A