PEPFECT

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Preparation of Stock Solution

1. Reconstitute one vial of PepFect in 500 µl Reconstitution Buffer (1 mM trifluoroacetic acid).

This yields a 100x stock solution.

2. Mix and vortex the vial with reconstituted PepFect to ensure that the peptide is entirely dissolved.

3. Store dry PepFect and its stock solution at –20°C.

Transfection Procedure

These instructions are for transfecting cells that are seeded in 24-well tissue culture plates (surface area 2 cm2). For transfections in tissue culture plates with a surface area other than 2 cm2 the volumes and amounts should be adapted accordingly.

For transfecting 1 well of a 24-well tissue culture plate do the following:

DAY 0

Seed cells into a 24-well tissue culture plate in 450µl of normal growth medium per well.

DAY 1

Note: Prepare PepFect complexes in 1/10th of the final transfection volume.

The final transfection volume after adding the complexes will be 500 µl per well.

In volume of 50 µl OptiMEM, mix 50 pmol antisense oligonucleotide (AON) or 10 pmol siRNA or 0.25 µg plasmid DNA with 0.5 µl reconstituted 100X stock solution of PepFect.

Incubate the mixture at room temperature for 30 min to allow complex formation.

Add 50 µl of the formed PepFect complexes to cells grown in 450 µl of normal growth media.

Note: the final concentration of the added oligonucleotides will be 100 nM AON, 20 nM siRNA and 0.5 µg/ml plasmid DNA.

After 4 hours, remove the added complexes and replace with fresh cell growth media.

DAY 2

24 hours after adding the complexes, measure the activity of the transfected AON, siRNA or plasmid.

Note: The amount of antisense oligonucleotide, siRNA and plasmid DNA can be varied to optimise the transfection conditions depending on application.