SEAP Expressions in BHK21 Cells After Transfection with FuGENE®HD, FuGENE®6 and competitors

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Transfection with competitor reagent was performed according to manufacturer’s instructions

SEAP BHK21 FuGENE HD, FuGENE®6 and competitors comparison

1. Cell plating

   BHK21 cells were seeded from 90-100% confluent culture the day before transfection with the density 10,000 cells per well in 100 μl complete growth medium DMEM + 10% Fetal Bovine Serum (Gibco®).

2. Complex preparation (per 20 wells)
   Tissue culture 96-round bottom well plates were used for complex preparation:
   1. Prepare 0.02 μg/μl gWIZ™ SEAP DNA solution in Opti-MEM®I or sterile
      deionized water.
   2. Add 6 μl of FuGENE®HD to 100 μl of DNA solution.
   3. Mix carefully by pipetting (10-15 times).
   4. Incubate 5 minute at room temperature.
   5. Add 5 μl of complex per well to the cells, and mix thoroughly.

   Optimal ratio of reagent/DNA varies from 0.25μl/0.1μg to 0.4μl/0.1μg.

3. Incubation
   Place the cells into CO₂ incubator for 26-28 hours. Monitor the transgene expression.
4. Detection of SEAP expression

   SEAP activity is quantitatively determined by a colorimetric method based on hydrolysis

   of chromogenic substrate para-nitrophenyl phosphate (PNPP). 1 μg/μl of PNPP is

   prepared in 1M Diethanolamine, 1mM MgCl₂, pH9.8.

   1. Transfer 5 μl of transfected cells supernatant into a well of flat bottom 96-well plate.
   2. Add 100 μl of PNPP reagent.
   3. Incubate for 5 min at room temperature in the dark place.
   4. Read the plates at 405 nm.