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

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QUOTE (MCR @ Jul 25 2006, 07:02 PM)
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.

Your cells should be seeded in normal culture medium with antibiotics omitted. Reduced serum medium (Opti-MEM) is only used to prepare the transfection complex.


Opti-MEM really does work better. I greatly improved my transfection efficiency once I finally decided to buy Opti-MEM rather than just the DMEM I had lying around. My colleague used to work with liposomal formulations in industry and said that they are very sensitive to certain salt concentrations etc.. thus it's best to work with what Invitrogen recommends.


DMEM is worse than RPMI, but Opti-MEM is indeed best for transfections.


Thanks i had a very nice transfections!!!!!!

QUOTE (pcrman @ Jul 25 2006, 01:36 AM)

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.


hai....there...this is my first time i make transfections...actualy i was give a task which i must make expression gene on cell hamster ovary or NSO cell as a host ( how about retrovirus or pcDNA 2.1).so i'm thinking of what vector should i use to make it happen...can somebody out there give sugestion what compatible vector can be use and other information which related with that....thanks


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