Sure! Here's a transfection protocol for HEK-293 (https://altogen.com/product/hek-293-transfection-reagent-epithelial-kidney-cells/). It can be scaled down to 6 wells, and here's the protocol for that (you may want to adjust it for efficiency purposes):

1. Plate 60,000 - 90,000 HEK293 cells per well in 3 ml of

complete growth medium 12–24 hours prior to transfection

2. Wash with 1xPBS and add 3 ml of fresh growth medium

3. Prepare transfection complexes by mixing 240 µl of serumfree

medium, 30 µl of transfection reagent, and

• 500 ng DNA (or mRNA), or

• 30 nM - 50 nM of siRNA (or microRNA)

4. Incubate transfection complexes at RT for 15 - 30 minutes

5. Optional: Add 12 µl of Complex Condenser. This reagent

reduces the size of transfection complex, therefore increasing

transfection efficiency; however it may increase cell toxicity

6. Add prepared transfection complexes to 3 ml of

complete growth medium with HEK293 cells (from step 2)

7. Incubate cells at 37ºC in a humidified CO2 incubator

8. Assay for phenotype or target gene expression 48 - 72

hours after transfection

You should be able to. However, it is always best to optimise the amount of DNA you use for transfection.  I used the amount of DNA between 0.25-2.5 ug and found little difference and didn't observe any toxicity.  But of course it depends on your ultimate results (I.e. What are transfecting your cells for) . Hope it helps

Dear Sleman,

Depending on several parameters (DNA, promoter, transfection reagent, cell clone, medium, tume of transfection, level of confluence, washing or not after transfection, ...) 2 µg may be toxic ; moreover, in general, transfection reagent volume directly depends on DNA amount. And DNA + Transfectant can be toxic.

The first thing to do, is to settle conditions with a control plasmid encoding for a control gene such as GFP or LacZ (you can, for example use any of these positive controls plasmids : http://www.ozbiosciences.com/transfection-controls/55-plasmids-pvectoz.html).

This will allow you :

• finding ideal conditions for the transfection and then apply these conditions to your plasmid of interest
• finding if yuor transfection system is toxic (association of plasmid + transfection reagent).

If there is still toxicity, I would definitively recommend you a transfection reagent that is based on biodegradable properties that presents a high level of nucleic acid compaction and that work very well for any cell lines of whom HEK-293: DreamFect Gold.

In the attached file you can find a sample list of publication mentionning the use of DreamFect Gold to transfect DNA into HEK-293, and for more publications you can refer to : http://www.ozbiosciences.com/module/citationfinder/default

I would be really glad to send you a sample to test, you can contact me directly at [email protected]

I hope this will be of help,

good luck with your experiment,

best regards,

Cedric

Thank you Olga ... do you use also PEI reagent ?? 

We routinely use Lipofectamine 2000 when transfecting 293 cells. I would suggest optimizing you transfection conditions.  We have found that removing the transfection media and replacing after 4-8 hrs can help increase the cell viability.  

I prefer to use lipofectamine plus

I was using Lipofectamine 2000 (life technology) when transfecting HEK 293 and HEK 293T cell line. As suggested by Jonathan, remove transfected media after 6 hrs of transfection will definitely increase the cell viability.

of course depending on your yield....but i transfected HEK293 cells in a 6-well plate with 1µg DNA ...and found that it's sufficient...i think important is also that your cells are confluent enough...if there are too few...it's more toxic...I transfected with EcoTransfect (from OZBiosystems) in a 1:2 ratio (DAN/Transfection reagent) ...the medium change helps...

I found these cells really easy to transfect with FuGene6 reagent. Confluence was at about 50% in the 6-well and I used a 3:1 ratio of reagent:DNA (i.e. 47ul media + 3ul reagent + 1ug Clontech expression vector (e.g. pSomething-EGFP)). I didn't optimise but I feel the quantity of DNA could be taken down. The transfection media was left on overnight with no obvious toxicity.

removing the transfection media after 6 hours might increase the cell viability but will give very little expression. Time course also needs to be optimised.

Hi Selman,

I have regularly been able to transfect HEK cells in 6 well plate with 3ug DNA. I use 3ul fugene or 6ul BioT transfection agent. Make sure you use endofree maxi prep plasmids. 

Thanks Kasturi 

I have compared the transfection efficiency with Lipo and PEI and found that PEI work just as well if not better than Lipo.  I have transfected ~3 ug in total in 35 mm plates and got ~80% transfection efficiency

I usually transfected my HEK239T overnight and change the medium next morning, since PEI is less toxic to cell compare to Lipo, I don't see as much cell death with PEI compare to Lipo.

Thanks Kevin ,

in which ratio you use PEI reagent  1:4 ?? 

and your PEI is self made ?? PEI 1ug/ul  from sigma from Polysciences (cat# 23966–2) or from polyplus ?? 

THANKS 

I use 3:1 ratio, I just made up mine in 1mg/ml, pH 7 stock and keep at 4 degree, we got our from Polysciences (#23966 2 g).  I have used Fugene, Lipo, Neon and now PEI, in my opinion PEI is by far the cheapish alternative, especially since we do large scale transfection.

Thanks Kevin 

so i can to do co-transfection of  two plasmid 1ug each one (2ug total) using PEI ?

many Thanks for your help 

sorry I have no experience

Hi Sleman, Yes you can.  I transfected mine with a combination of 4 plasmids, total of ~3 ug DNA.  You already know this, but make sure to use antibiotic free media during the transfection.  Its pretty straight forward.  Let me know how it go.

Kevin Ly many thanks 

I am planing to do lipo transfection of HEK293 cells in 6 wells plates.I will use 5ug of my DNA to each well of the plate.Is there any one who can share me his/her success experience? Many thanks in Advance.

Hello Anteneh,

I regularly transfect 4ug DNA per well of a 6 well plate in HEK293 cells and it works fine. You should not have any problems

 Many thanks Kasturi Pal

What volume of Serum free medium you were used per each well?

Hi Selman

I always transfect HEK cells in 6 well plate with 5ug DNA with 50-60%confluence.I used lipofectamine 2000 as transfection reagent.The result was interesting.

Hope hear you soon

Best

Anteneh

Hi Selman,

We are using DreamFect Gold or Ecotransfect to transfect HEK cells in 6 well plate with 1µg DNA

http://www.ozbiosciences.com/transfection-dna/19-dreamfect-gold-transfection-reagent.html

If you want free samples of these reagents let me know at [email protected]

Good Luck

Hi.  2ug should be fine.  You should expect to see 80-90% transfection efficiency with PEI.  I use serum free medium during the tranfection and replaced with full complete medium after 6 hr.  Not sure about 5ug thought that's a lot of DNA for the volume of cells.

Sure! Here's a transfection protocol for HEK-293 (https://altogen.com/product/hek-293-transfection-reagent-epithelial-kidney-cells/). It can be scaled down to 6 wells, and here's the protocol for that (you may want to adjust it for efficiency purposes):

1. Plate 60,000 - 90,000 HEK293 cells per well in 3 ml of

complete growth medium 12–24 hours prior to transfection

2. Wash with 1xPBS and add 3 ml of fresh growth medium

3. Prepare transfection complexes by mixing 240 µl of serumfree

medium, 30 µl of transfection reagent, and

• 500 ng DNA (or mRNA), or

• 30 nM - 50 nM of siRNA (or microRNA)

4. Incubate transfection complexes at RT for 15 - 30 minutes

5. Optional: Add 12 µl of Complex Condenser. This reagent

reduces the size of transfection complex, therefore increasing

transfection efficiency; however it may increase cell toxicity

6. Add prepared transfection complexes to 3 ml of

complete growth medium with HEK293 cells (from step 2)

7. Incubate cells at 37ºC in a humidified CO2 incubator

8. Assay for phenotype or target gene expression 48 - 72

hours after transfection

Hi, i want to do transfection for the first time by using HEK293T cells, I want to know whats the difference between HEK293 and HEK293T cell or they are same? And how much cells i have to add per 6 well plate to get better transfection efficiency?