Hi Mansi

In my experience, it is appropriate to mix the lipo and DNA firstly, then the mixture should be added drop by drop into the media. Change the media before transfection would be very helpful.

Genemedi launched a product of Lipogene, comparable to lipofectamine 3000. You could find more information on this website: www.genemedi.net/i/lipogene-transfection-reagent

What we usually do is We make a master mix of DNA and Lipofectamine, incubate it and add it to the media directly. After 4-6 hours we refresh the media. I don't think it is a wise idea to do after removing the media. Removing the media might stress the cells and result in cell death. 

Thank you Chandhuru, I will try adding it directly to media. And in your case does it affect the transfection effeciency?

Hi Mansi,

I agree with Chandhuru, in that you should be adding your DNA and lipofectamine mixture directly to the media in a dropwise fashion.  However, I would like to add a couple of points that help minimize toxicity and have boost transfection efficiency in our hands:

This is what works best in our lab.  Anyway, I hope this helps.  Transfection can be tricky, especially if you have a fussy cell line.  Good luck and keep at it!  All the best.

--Bryan

Thank you Bryan for suggestions, will definitely implement them.

Hi Mansi, 

This is the protocol I use for primary fibroblast, MAC-T and HEK 293 cells. I usually get between 60-80% transfection efficiency.

For transfecting  cells in 6 well plates with 2µg of plasmid DNA using Lipofectamine 3000. Enough for 1 well

1.    Plate down cells up to 90% confluency 

2.    Gently wash with 1ml of prewarmed DPBS and replace media with opti -MEM (30min - 1 h before transfection)

3.    Warm all reagents to room temperature

4.    Make two 125µl aliquots of opti-MEM ( A and B)

5.     To tube A add  6µl of lipofectamine 3000 (1µg DNA : 3µl ) vortex briefly and spin down

6.     To tube B add 4µl of P3000 reagent (2µl :  µg DNA ) vortex briefly and spin down

7.    Add add all the contents of tube B to Tube A  mix by pipetting brifelly. 

8.    Incubate for 5 minutes

9.    Add in small drops to your well 

10.  Change the opti-MEM for DMEM-F12 10% FBS 12h after transfection.

Hi Carlos

Thanks for your protocol but I have a doubt. In which tube do you put your DNA? A or B?

Thanks

I was wondering the same Pablo, I think, tube A refers to lipo-DNA complex mix. So you put your DNA in tube A

Tube A 125 uL of OptiMEM => x ug of DNA + 3x uL of  lipofectamine

Tube B 125 uL of OptiMEM => 2 uL of P300 REAGENT

Vortex both tubes after addition

Mix B into A

Incubate 5' (I like to do it at least 20')

Add it to your cells

If I got wrong by any chance, please correct me! :) 

Hey, David, I tried the protocol and I asked  the people from Thermo and the order is 

Tube A: DNA + P3000 reagent

Tube B Lipofectamine

I followed  the protocol from Carlos and it works:D

Hello I'am working on transfection of shrimp sperm. Is it necessary to add opti-MEM using lipofectamine 3000?

Hi Doris,

I don't know which media is used for shrimp cells but we use serum free media for our experiment. do not use serum containing media neither for DNA:lipo mix preparation nor initial 4-6 hours incubation . It's really minimize the transfection efficiency.

A lot of the transfection protocol/efficiency depends on the kinds of cells you are using and what kind of effect you are needing. For stable transfections, lipofection-based approaches can work with minimal cytotoxicity for siRNA transfection (see https://altogen.com/product/altofect-transfection-reagent/), but for larger plasmids it may be more difficult. When I do lipofection, I add complexes directly to the medium, and that generally seems to keep cells healthy and result in good efficiency.

Carlos Andres Pinzón

Hi Carlos, may I ask about the software name that for counting the cell number? thanks a lot.

I find the protocol for lipofectamine 3000 very confusing with the 2 reagents that have a very similar name! on top of that the insert shows an example for 2 wells instead of one. I corrected the insert and attach it here. Other important considerations: use medium with regular amount of serum (removing the serum was needed for older version of the transfection reagents and is a tradition that should be discarded) but w/o antibiotics. If you use medium with antibiotic you will have more cell mortality but that is not a problem with low amount of DNA (you can check this in your test experiment to determine the best conditions). I normally transfect late so I change back to regular medium the next morning. An other important point: Lipofectamin is a lipid and the incubation periods are there to allow to form lipid droplets containing DNA, therefore mixing of the reagent stock tubes and DNA-reagent mix has to be gentle but throughly.

Hi all, I am transfecting Drosophila S2 cells for Luciferase assay. My cells are in 24 well plates, in 1ml S2 complete media (10% FBS + antibiotics). Is it okay if I just add DNA-Lipid complex (50ul) to cells directly ? Or do I need to wash and replace the cells with 1ml serum free media before adding the DNA-Lipid complex ?

(since Invitorgen claims transfection works with media containing serum and antibiotics with Lipofectamine 3000)

Hi Mansi

In my experience, it is appropriate to mix the lipo and DNA firstly, then the mixture should be added drop by drop into the media. Change the media before transfection would be very helpful.

Genemedi launched a product of Lipogene, comparable to lipofectamine 3000. You could find more information on this website: www.genemedi.net/i/lipogene-transfection-reagent

What is the time required for incubation for luciferase after transfection with lipofectamine 3000?

Hi Soledad Miranda-Rottmann ,

I am trying to optimize transfection using Platinum E cells. Can i use Pcl-ECO as a packaging vector and P3000 provided with the Lipofectamine3000 kit to improve my transfection and get higher viral titres? Also, I would like to make virus in bulk as I run out of them pretty often. Can you suggest any recommendations to make large batches of virus?

Vineet Garlapally You would have to try. As far as I know the composition of the P3000 as not been disclosed, so all bets are open. It might just be useful for the Lipofectamine 3000. Maybe is just a buffer to adjust the pH of the solution.