7 replies to this topic

[Mark10293]

Hey I was wondering if anyone could help me who has used the standard lipofectamine protocol before. Do you remove all media from your wells before adding DNA complexes, or do you leave it in?


I am using a 24 well format, so I grow my cells in about .5ml of media in each well. Am I supposed to leave it or remove it and just add the ~100 ul of lipofectamine reagents by itself?

Thanks.

[lotus]

serum has substances that will inhibit the lipofectamine. so the medium has to be removed and the cells washed with PBS to remove all traces of serum. i used to use lipofectamine 2000 with either optimem or plain dmem with no additives.

[WOW]

lotus, on Mar 20 2009, 05:17 AM, said:

serum has substances that will inhibit the lipofectamine. so the medium has to be removed and the cells washed with PBS to remove all traces of serum. i used to use lipofectamine 2000 with either optimem or plain dmem with no additives.

If you could not acheive ideal transfection efficiency, you'd better do it as the manufacturer suggests. However, according to my experience in my cell (Ishikawa, a cancer cell line), serum had no effect to lipo-transfection. My protocol is 1ug DNA+2ul lipo in 200 ul opti-mem for 12 well dish, 800ul culture medium per well. Nearly 100% efficiency. May it help you and good luck.

[MaggieRoara]

WOW, on Mar 19 2009, 10:00 PM, said:

lotus, on Mar 20 2009, 05:17 AM, said:

serum has substances that will inhibit the lipofectamine. so the medium has to be removed and the cells washed with PBS to remove all traces of serum. i used to use lipofectamine 2000 with either optimem or plain dmem with no additives.

If you could not acheive ideal transfection efficiency, you'd better do it as the manufacturer suggests. However, according to my experience in my cell (Ishikawa, a cancer cell line), serum had no effect to lipo-transfection. My protocol is 1ug DNA+2ul lipo in 200 ul opti-mem for 12 well dish, 800ul culture medium per well. Nearly 100% efficiency. May it help you and good luck.

Lipofectamine 2000 doesnt seem to be hindered by serum or antibiotics for me.

[cotchy]

Same here, lipofectamine2000 had no problem functioning in the presence serum when using it to immortalise a primary cell line.

As an  aside, further transfections on the now immortalised cells using lipofectamine2000 (same conc DNA and lipo as first transfection) in the presence of serum has not shown any uptake of plasmid


Cotchy.

[Dr Teeth]

Lipofectamine is hindered by the presence of serum.  Lipofectamine 2000 is not.

[scolix]

I change the media to one without antibiotics before adding the lipofectamine mixture.

[Mark10293]

Thanks for the replies everyone, but now I have another problem. When I put in the serum free medium, a large number of my cells will die. I have remade the medium, and still the same problem. Has anyone else run into this?