How do you transfect siRNA?

How do you transfect siRNA?

9 Tips for Optimal siRNA Transfection

  1. Use the most appropriate siRNA concentration.
  2. Prepare a suitable siRNA stock solution.
  3. Transfect healthy cells.
  4. Check serum quality.
  5. Know the target gene in and out.
  6. Always use positive and negative controls.
  7. Follow up the transfection reagent protocol.

What is Accell siRNA?

Guaranteed gene silencing in difficult-to-transfect cells. A novel siRNA for difficult-to-transfect cells, modified to require no transfection reagent or viral vector for delivery. Predesigned Accell siRNA is available as individual reagents and in SMARTpool format.

What is DharmaFECT?

DharmaFECT is the only transfection reagent offered in four distinct siRNA-specific formulations to ensure optimal siRNA delivery in a wide range of cell lines. No single lipid reagent outperformed the panel of DharmaFECT formulations in a. benchmark study of twenty common cell lines.

How much siRNA do you use for transfection?

As mentioned above, too much siRNA may lead to off-target effects; too little can result in undetectable gene silencing. In general, 1-30 nM siRNA is a good concentration range within which to optimize transfection (10 nM is a sufficient starting point).

How do you dissolve siRNA?

General considerations: Dissolve siRNA in sterile water and prepare 5 µl aliquots, store at –80°C. For use, dilute siRNA in siRNA dilution buffer (diluted siRNA is not stable, use only once). 5x siRNA buffer aliquots (Dharmacon) are stored at 4°C in the cell culture lab.

How do you optimize a siRNA transfection?

Tips for a successful siRNA experiment

  1. Design and test two to four siRNA sequences per gene.
  2. Avoid RNases!
  3. Maintain healthy cell cultures and strict protocols for good transfection reproducibility.
  4. Avoid antibiotic use.
  5. Transfect siRNAs using optimized reagents.

How do you dilute siRNA?

To dilute the 5x siRNA Buffer to 1x siRNA Buffer, mix four volumes of sterile RNase-free water with one volume of 5x siRNA Buffer. The composition of the 1x siRNA Buffer is 60 mM KCl, 6 mM HEPES-pH 7.5, and 0.2 mM MgCl2.

How do you dilute dharmacon siRNA?

How do you make siRNA?

Currently, there are five methods for generating siRNAs for gene silencing studies:

  1. Chemical synthesis.
  2. In vitro transcription.
  3. Digestion of long dsRNA by an RNase III family enzyme (e.g. Dicer, RNase III)
  4. Expression in cells from an siRNA expression plasmid or viral vector.

What is Lipofectamine reagent?

Lipofectamine or Lipofectamine 2000 is a common transfection reagent, produced and sold by Invitrogen, used in molecular and cellular biology. It is used to increase the transfection efficiency of RNA (including mRNA and siRNA) or plasmid DNA into in vitro cell cultures by lipofection.