help needed: reagent... PEI protocol

Juha Saarikettu juha.saarikettu at cmb.umu.se
Fri Nov 23 02:57:43 EST 2001


About transfection reagents.
I am using PEI filtered through Amicon YM-10 and it works fine for at
least plasmid DNA. I haven't done comparisions between filtered and
non-filtered PEI on cytotoxicity but I have a feeling that transfection
efficiency is better with the filtered PEI. Probably there is a
commercial low MW PEI.

Polyethylenimine (PEI) transfection

PEI  is made from the 50% aqueous solution (SIGMA #P-3143). 100 mg of PEI
in 1,8 ml H20, let dissolve O/N at +4C (rotation) and add 130 ul 5M HCl.
The final pH is around 6. Sigma PEI is a mixture of different molecular
weight polymers. It has been shown that short  (<10 Kda) PEI is better for
transfections and has low cytotoxicity.
Filter the dissolved PEI through Amicon YM-10 filter (Millipore).

IMPORTANT: the optimal DNA/PEI ratio between different PEI filtrates
varies significantly and has to be titrated for each new filtrate.

1. Seed the fibroblast cells 300 000 cells/5 cm plate 16-24 h before
transfection.
2. Just before transfection change the medium to 1 ml serum free medium.
OPTI-MEM or normal medium with all the additives except serum can be used.

3. Make the following dilutions:

DNA TUBE:
MIX 4 ug of your DNA with 0,5 ml serum free medium.

PEI TUBE:
MIX 6 ul (or the optimised amount) of PEI  with 0,5 ml serum free medium

5. incubate the tubes 5 min RT and spin
6. Dropwise add the PEI solution from the PEI tube to the DNA tube, mix by
inverting the tube 10 times and  incubate 10 min RT.
7. Centrifuge down the drops from the lid and add the PEI/DNA on cells,
incubate 2-6 hours.
8. Change the medium to serum containing medium.

Proc Natl Acad Sci U S A 1995 Aug 1;92(16):7297-301
A versatile vector for gene and oligonucleotide transfer into cells in
culture and in vivo: polyethylenimine.

Biotechniques. 2001 Jan;30(1):74-7, 80-1
Preparation of a low molecular weight polyethylenimine for efficient cell
transfection.


Juha Saarikettu
Umeå University
Sweden


David L Haviland wrote:

> Wolfgang:
>
> > Or she just might try polyethyleneimine (PEI) transfection. works
> > on many
> > cells and is cheap (1 bathtube full of reagent for almost no cost).
>
> Where are these?  I'm interested in trying PEI but heard that toxicity
> is a problem but I am tempted given the price.
>
> Many thanks in advance,
> David
>
> ============
> David L. Haviland, Ph.D., Asst. Prof. Immunology
> University of Texas - Houston, H.S.C.
> Institute of Molecular Medicine, R907
> 2121 W. Holcombe Blvd.,  Houston, TX  77030
> 713.500.2413-Voice//713.500.2424-FAX
> -----------------
> If everything seems to be going so well, you have obviously
> overlooked something.
> ============
>
> ---
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