Stealth RNAi™ Resuspension & Cloning Protocols
Storage and stability of Stealth RNAi™ siRNA or siRNA:
Store as a dry pellet at –20°C until ready to use. Once resuspended, store at –20°C and avoid contact with RNAses. As a dry pellet the RNA is stable at least 6 months.
Resuspending Stealth RNAi™ siRNA or siRNA
Resuspend siRNA or Stealth RNAi™ siRNA duplexes in DEPC-treated water according to the chart in order to make a 20 µM solution. The RNA oligo was dried down from a buffered solution. Resuspension to 20 µM will reconstitute the buffer to 10 mM Tris-HCl, pH 8.0, 20 mM NaCl, 1 mM EDTA.
|Delivered Quantity/Purity||Resuspension Protocol|
|20 nmole desalted||Resuspend the 20 nmole yield in 1000 µl RNase-free water in order to make a 20 µM solution.|
|80 nmole desalted||Resuspend the 80 nmole yield in 800 µl RNase-free water to make a 100 µM solution. Dilute 1:5 to create a 20 µM working stock.|
|1.0 µmole desalted||Resuspend the 1 µmole yield in 1 ml Rnase-free water to make a 1mM solution. Dilute 1:50 to create a 20 µM working stock.|
|1.7 µmole desalted||Resuspend the 1.7 µmole yield in 1.7 ml Rnase-free water to make a 1mM solution. Dilute 1:50 to create a 20 µM working stock.|
Stealth RNAi™ siRNA and siRNA transfection concentrations
The transfection concentration of a Stealth RNAi™ siRNA or siRNA duplex is determined by dividing the molar amount used by the final volume of the transfection (i.e., starting medium volume + transfection mixture volume). With a potent siRNA, we typically transfect 0.5 – 5 pmol by adding 100ul transfection mix to 500ul medium (0.8 - 8nM final concentration) per well in a 24-well plate. To scale up, increase the amount of siRNA to keep the concentration the same. We recommend using the minimum amount of siRNA that will knockdown your gene to avoid any non-specific or off-target effects.
Storage and Stability of Annealed dsDNA
Store as a dry pellet at –20°C until ready to use. Once resuspended, store at –20°C for at least a year.
Resuspending DNA oligos for cloning RNAi vectors
The single-stranded oligos are supplied lyophilized. Immediately before use, resuspend the single-stranded oligos in water or TE Buffer to a final concentration of 200 µM before use. Anneal equal amounts of the top and bottom strand DNA oligos to generate the ds oligos. We perform the annealing reaction at a final single-stranded concentration of 50 µM. Annealing at concentrations below 5 µM significantly reduces the efficiency. Note that the annealing step is not 100% efficient. To clone the ds oligo into the RNAi vector dilute the 50 µM stock to a final concentration of 10 nM and then proceed to clone this into the RNAi vector and transform your ligation reaction into competent TOP10 E. coli.