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BioT plasmid transfection protocol

Using the following procedure to transfect DNA into mammalian cells in a 35 mm 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. Prepare complexes using a BioT (µl) to DNA (µg) ratio of 1.5:1 for most cell lines or primary cells. Optimization may be necessary.

  1. Grow your cells to 60 –75% confluence in a 35 mm culture dish with 1.5 – 3 ml growth medium (any growth medium).
  2. For each transfection sample, prepare the transfection complexes as follows: In a sterile 1.5 ml microtube, mix the following reagents: 

    100 µl
    Serum-free DMEM (or any other serum free-medium, PBS, DPBS etc.
    Please do not use OPTI-MEM!
    2 µg Plasmid DNA
    3 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 35 mm culture dish. Tilting the dish a few times to mix. Return the dish to a CO2 incubator. Serum concentration in the growth medium has not effect on the transfection efficiency. Up to 20% of fetal bovine serum has been tested without significantly changing the transfection efficiency. 

  3. 16 to 24 hours after transfection, replace the medium in the dish with 2 ml (or any volume appropriate) fresh growth medium. Note: If no toxicity is observed, change of medium is not needed. If high toxicity is observed, either adjust the amount of DNA and/or shorten incubation time to 5-8 hours.

  4. Protein expression should reach a high level at or after 48 hours post-transfection.

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

 

Culture dish

Surf. Area per well (cm2)

Shared reagents

DNA transfection

Vol. Of Plating Medium

Vol. Of mixing Medium

DNA

BioT

10 cm

56

10 ml

500 µl

10 µg

15 µl

60 mm

21

3 ml

150 µl

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