9 replies to this topic

[goldfinger]

Hi all, usually the manual and protocols come with transfection reagents will tell you to try out a best DNA/reagent ratio for success transfection. But I was wondering about how much media should be used to make the transfection complex, say how much FBS free media should I use to mix with 3ul transfection reagent? 100ul? what about 30ul transfection reagent then? 1ml? is this a fixed ratio or we need to try out too? I worry that too much FBS free media will dilute the FBS concentration in the cell culture and who know what consequence it will has.
    Also, is there any requirement for what media should be used to make the transfection complex? Is DMEM good enough or you have to use Opti-MEM? How much difference can there be?

[bob1]

Both fugene and lipofectamine, and their derivatives tell you how much medium to dilute the reagents in.  Basically it works out to be the half the volume that you would normally put into a well of a plate (for any plate size), then add to cells in serum containing medium making up the other 50% of the volume.

[laurequillo]

bob1, on Feb 24 2010, 12:07 AM, said:

Both fugene and lipofectamine, and their derivatives tell you how much medium to dilute the reagents in.  Basically it works out to be the half the volume that you would normally put into a well of a plate (for any plate size), then add to cells in serum containing medium making up the other 50% of the volume.

That´s right. Normally we use 50ul(12 wells/plate), 100ul(6 wells/plate) or 200ul (10cm dishes)

[goldfinger]

laurequillo, on Feb 24 2010, 12:23 AM, said:

bob1, on Feb 24 2010, 12:07 AM, said:

Both fugene and lipofectamine, and their derivatives tell you how much medium to dilute the reagents in.  Basically it works out to be the half the volume that you would normally put into a well of a plate (for any plate size), then add to cells in serum containing medium making up the other 50% of the volume.

That´s right. Normally we use 50ul(12 wells/plate), 100ul(6 wells/plate) or 200ul (10cm dishes)

so, when you transfect with 3ul or 20ul reagent in 10cm dishes, the media volume is always the same 200ul?

[laurequillo]

goldfinger, on Feb 24 2010, 04:05 PM, said:

laurequillo, on Feb 24 2010, 12:23 AM, said:

bob1, on Feb 24 2010, 12:07 AM, said:

Both fugene and lipofectamine, and their derivatives tell you how much medium to dilute the reagents in.  Basically it works out to be the half the volume that you would normally put into a well of a plate (for any plate size), then add to cells in serum containing medium making up the other 50% of the volume.

That´s right. Normally we use 50ul(12 wells/plate), 100ul(6 wells/plate) or 200ul (10cm dishes)

so, when you transfect with 3ul or 20ul reagent in 10cm dishes, the media volume is always the same 200ul?

Well, normally we use 8-20 ul of the reagent (It depends of the amount of plasmid we want to transfect, normally between 3-10ug) in 200ul for 10cm dishes

[goldfinger]

laurequillo, on Feb 24 2010, 10:09 AM, said:

goldfinger, on Feb 24 2010, 04:05 PM, said:

laurequillo, on Feb 24 2010, 12:23 AM, said:

bob1, on Feb 24 2010, 12:07 AM, said:

Both fugene and lipofectamine, and their derivatives tell you how much medium to dilute the reagents in.  Basically it works out to be the half the volume that you would normally put into a well of a plate (for any plate size), then add to cells in serum containing medium making up the other 50% of the volume.

That´s right. Normally we use 50ul(12 wells/plate), 100ul(6 wells/plate) or 200ul (10cm dishes)

so, when you transfect with 3ul or 20ul reagent in 10cm dishes, the media volume is always the same 200ul?

Well, normally we use 8-20 ul of the reagent (It depends of the amount of plasmid we want to transfect, normally between 3-10ug) in 200ul for 10cm dishes

Thanks! one more question, what media you use for reagent dilution? I use DMEM for cell culture and reagent dilution, but I heard OPTI media is better for reagent dilution, what you think?

[Dr Teeth]

goldfinger, on Feb 24 2010, 11:43 AM, said:

laurequillo, on Feb 24 2010, 10:09 AM, said:

goldfinger, on Feb 24 2010, 04:05 PM, said:

laurequillo, on Feb 24 2010, 12:23 AM, said:

bob1, on Feb 24 2010, 12:07 AM, said:

Both fugene and lipofectamine, and their derivatives tell you how much medium to dilute the reagents in.  Basically it works out to be the half the volume that you would normally put into a well of a plate (for any plate size), then add to cells in serum containing medium making up the other 50% of the volume.

That´s right. Normally we use 50ul(12 wells/plate), 100ul(6 wells/plate) or 200ul (10cm dishes)

so, when you transfect with 3ul or 20ul reagent in 10cm dishes, the media volume is always the same 200ul?

Well, normally we use 8-20 ul of the reagent (It depends of the amount of plasmid we want to transfect, normally between 3-10ug) in 200ul for 10cm dishes

Thanks! one more question, what media you use for reagent dilution? I use DMEM for cell culture and reagent dilution, but I heard OPTI media is better for reagent dilution, what you think?

Opti-mem has always been better for me at least.

[laurequillo]

Yeah I used optimem with Lipofectamine and it worked quite well; another tip was that we did our mixtures in plastic tubes (the ones that you use for the luminometer or the FACS), and we got better results than using eppendorf.

Now I use other reagents and I just use DMEM without antibiotics and without serum for the mixture

[KevinK]

Goldfinger,
  The amound of media will depend on the number of wells you are transfecting.  Typically you will need to find the optimium DNA mass amount and lipid:DNA ratio for your cell line and reagent as well.  Some companies may provide a calculator on their website to help with this.  Promega recently released FuGENE® HD and we have a calculator that may be of use for this and other reagents: http://www.promega.c...tools/FugeneHD/

Hope this helps.

Kevin

[goldfinger]

KevinK, on Feb 24 2010, 05:24 PM, said:

Goldfinger,
  The amound of media will depend on the number of wells you are transfecting.  Typically you will need to find the optimium DNA mass amount and lipid:DNA ratio for your cell line and reagent as well.  Some companies may provide a calculator on their website to help with this.  Promega recently released FuGENE® HD and we have a calculator that may be of use for this and other reagents: http://www.promega.c...tools/FugeneHD/

Hope this helps.

Kevin

Thanks KevinK !
The charter really helps!