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BioT siRNA transfection protocol

Using the following procedure to transfect siRNA into mammalian cells in a 12-well dish. For other size of cell culture dish, refer to Table 1 for scaling up and down. All amounts and volumes are given on a per well basis. Optimization may be necessary.

  1. Split the cells one day before transfection to reach 70 – 80% confluence in the day of transfection.
  2. For each transfection sample, prepare the transfection complexes as follows: In a sterile 1.5 ml microtube, mix the following reagents (for 12-well dish):
50 µl  Serum-free DMEM (or any other serum free-medium, PBS, DPBS etc.) 

Please do not use OPTI-MEM!

40 pmol siRNA (2 µl x 20 µM siRNA)
1 µl   BioT
  1.  Pipetting up and down the mixture a few times. Spin briefly in a centrifuge. Leave the mixture at room temperature for 5 min.
  2. Add the entire mixture directly to cells in the 12-well culture dish. Tilting the dish a few times to mix. Return the dish to a CO2 incubator. Serum concentration (0-20%) in the growth medium has no effect on the transfection efficiency.
  3. Optional: At anytime between 5 to 24 hours after the transfection, replace the medium in the dish with 1 ml (or any volume you’d like) fresh growth medium. If toxicity is not observed, change of medium is not needed.
  4. Gene knockdown should be assayed 24-72 hours post-transfection.

  Table 1. Scaling Up or Down Transfections (based on plating medium volume)

Culture dish
Surf. Area per well (cm2)
Shared reagents
RNAi transfection
Vol. Of Plating Medium
Vol. Of mixing Medium
siRNA* (Range)
BioT
10 cm
56
10 ml
500 µl
400pmol (100-500pmol)
10 µl
60 mm
21
3 ml
150 µl
120 pmol (30-150pmol)
3 µl
35 mm
8
2 ml
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