First time doing transfections! - please!!! some help or tricks!!!! (Jul/24/2006 )

Hi all, i write because i need your help, i am going to do the first transfection in my life, the protocol that my P.I. gave me is using Lipofectamine 2000, i am new in this lab, and seems that he is having problems doing the transfections, i don't really know to much about this technique. so if someone have some ideas about this technique i really appreciate your help. The cell line that i am going to use for the transfection is 293 cells.

Thank you so much!!!

Hi MCR,

Transfection is not that hard to do. lipofectamine 2k is a good transfection reagent and you need to follow the protocol provided by invitrogen. Apart from that, you need to seed your cells at a proper density (about 50-60%, depending on your purpose). keep in mind that 293 cells are sensitive to lipofectamine. the transfection procedure is outlined below:

Day 0: Plate cells the day before transfection
Day 1:
1. dilute lipofectamine in Opti-MEM (Invitrogen) reduced serum medium to make solution A
2. diulte DNA or siRNA in Opti-MEM to make solution B
3. Combine A and B to let complex form
4. Add the mixture to your cells
Day 3 or 4: harvest cells for analysis

Hope that helps.

May I add that 293 line is very easy to transfect, and all the "cheap" methods e.g. CaPO4, PEI etc. will do the trick for this line. Don't forget to transfect with GFP in order to evaluate the transfection efficiency

I can give you some numbers: 200 000 cells in a 35mm dish; 2,4ug total DNA with 8ul Lip2000 or 1,2ug total DNA + 80 pmol siRNA with 8ul Lip2000 or 80pmol siRNA with 4ul Lip2000. You can use normal DMEM as well as OptiMEM.
As pcrman said you will make a mix A, that should incubate at RT for 5 min; when you mix A and B, you should incubate 20 min at RT and then add to the cells. If you transfect GFP, you can have a look at the fluorescence after 18 hours already.

i am also a beginner in this field and had no luck so far!!

i am trying to transfect 3 plasmids to Hela cells and and my reporter gene is SEAP,

My problem is the background SEAP level (very high) is same in my untransfected cells compared to positive and negative control. Just to give u some background...

1)I tried transfecting with and without serum,

2) and when after 5h i change the medium, I had tried using 5% and 10% serum with and without P/S

3) even after 2 days of transfection, i can see cells nicely adhered to the TC plate.

4)I am using Lipo 2000 and making my complex in opti-MEM.........As per the instructions I am using just 0.3 ug of each plasmid DNA as the total amt shd be 1 ug perwell in a 24 well plate,I was planning to run 0.3 ug DNA on gel exactly as i sometimes wonder as its too low!!!


I dont understand whats going wrong except that my my DNA preparations were not very clean as the 260/280 ratio was between 1.3-1.4. When i spoke to one of the tech support they say that ratio is not that critical when using Lipo 2000.

Can anybody suggest me any thing or please share your protocol ?

thanks ,
jhil

I somehow find suspension transfection using lipofectamine better.

Also its preferable to use clean DNA for transfections.

this may be a stupid question, but...why make solution A and solution B separately? if they're both being diluted in the same type of medium, why not just mix dna, lipofectamine, and medium together in one tube (this is how we do it in our lab, and it works)...is there a reason for diluting separately? or is it just more of a difference in technique (more careful, eg?)?

Hi,thanks for all your help, but i have some questions why you need to use Opti-MEM and not just MEM????, what is the importance in the concentration of serum during the transfection???? and why 293 cells are sensitive to lipofectamine

I am going to do my transfection on thursday, i have my Adeasy vector ready, so i hope that everything goes ok.

OPTIMEM (no serum) bcoz invitrogen suggests it when using lipofectamine.