What concentration of siRNA should I use?

What concentration of siRNA should I use?

In general, 1-30 nM siRNA is a good concentration range within which to optimize transfection (10 nM is a sufficient starting point). In Figure 6, transfection of HeLa cells was optimized at very low concentrations of siRNA.

How is siRNA concentration calculated?

What is your target concentration to treat the cells? Like, if you want to treat 100 nM concentration, the calculation will be ((100 nM/20 uM)*500 uL) = ((100 nM/20 x1000 nM)*500 uL) = 2.5 uL (of stock siRNA).

How do you dilute dharmacon 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.

Can I resuspend siRNA in water?

SiRNA buffer is recommended for long term storage, you can resuspend your SiRNA in RNAse free molecular grade water (or 0.22µm filtered DEPC treated water) for immediate use.

What is the concentration of siRNA for transfection?

1-30 nM
In general, 1-30 nM siRNA is a good concentration range within which to optimize transfection (10 nM is a sufficient starting point).

How can I improve my siRNA knockdown efficiency?

1. Be Consistent When Conducting Experiments.
2. Select Appropriate Order of Transfection.
3. Use Healthy Cells at the Optimal Density.
4. Choose the appropriate Culture Media and Culturing Conditions.
5. Use High Quality siRNA at the Lowest Effective Concentration.

How long does siRNA knockdown take?

5–7 days
Gene silencing resulting from siRNA can be assessed as early as 24 hours post-transfection. The effect most often will last from 5–7 days. However, the duration and level of knockdown are dependent on the cell type and concentration of siRNA.

How much siRNA do you use for transfection?

Can I Vortex siRNA?

Incubate the diluted siRNA mixture at room temperature for 5 min. Important: do NOT vortex the diluted siRNA mixture.

How do you optimize a siRNA transfection?

To optimize conditions, transfect target cells with several concentrations of an siRNA specific to your chosen positive control and to your experimental target siRNA. Measure the reduction in the control protein or mRNA level compared to untransfected cells 48 hours after transfection.

What is considered a good siRNA knockdown?

Generally, we see 95% or higher knockdown levels with our validated positive controls under optimized conditions. Efficiency below 80% indicates further optimization is needed. A non-targeting negative control siRNA to distinguish sequence-specific silencing from non-specific effects.

How long does it take for siRNA to work?

Gene silencing resulting from siRNA can be assessed as early as 24 hours post-transfection. The effect most often will last from 5–7 days. However, the duration and level of knockdown are dependent on the cell type and concentration of siRNA.