Transfection of Vero cells - Nothing new but it simply didn't work...... :( (Sep/12/2007 )
I am very new in transfecting plasmid DNA into mammalian cells. After my first transfection failure, I have read through most of the posts in this forum and took some of the advices into consideration when I did my next transfection. However, transfection still didn't work.
For my very first set of transfection, I followed strictly the protocol by Invitrogen. I did my transfection in 6-wells plate.
After reviewing the comments posted in this forum, I modified the protocol based on the suggestions that I felt reasonable and workable. Basically, I diluted my plasmid DNA and lipofectamine in 100uL OptiMEM. Incubated them at room temperature for a few minutes then added the diluted lipofectamine to the diluted plasmid DNA, incubated for 30 minutes at room temperature for the complex formation. Next, I diluted the complex mixture with OptiMEM to 1000uL and added 500uL to my Vero cells. 5-6 hours later, I added 5% FBS to the media and harvested the cells at 24 and 48 hours.
In one of the experiments, I removed the media and directly added 1000uL of the complex mixture to the cells, hoping that the cells would have direct contact to the lipid-DNA complexes.
I have played around with different amount of DNA and volume of lipofectamine ratio.
After all these experiments, nothing worked. I couldn't detect any product from my Western blot.
Can someone give me some suggestions and advices on this transfection?
Could it be my Vero cells are too old for transfection?
Or other transfection reagents work better for Vero cells?
Will electroporation work better despite of lots of cells death?
Many thanks in advance.
Dosages of DNA and Lipo2K, cytotoxicity and cell density are the key parameters to look upon.
Tell us more about what have you used and what have you observed.
I would certainly go with a repoter first before jump right into western.
I would recommend you to use smaller size multiwell plate to do an optimization work to save tiem and reagent.
In experimental details, I would not wait for 30 min, 5 min is enough, and I would add DNA dilution to lipo2K dilution instead.
Tell us more about what have you used and what have you observed.
I would certainly go with a repoter first before jump right into western.
I would recommend you to use smaller size multiwell plate to do an optimization work to save tiem and reagent.
In experimental details, I would not wait for 30 min, 5 min is enough, and I would add DNA dilution to lipo2K dilution instead.
Thank you, genehunter-1.
In the experiment where I added 1mL of the DNA-lipid complex to the cells after removing the media, for the well where I have used 4ug and 10uL lipofectamine (this is the maximum according to the protocol), I could observe quite a lot of cell death in that well. Other than that well, I couldn't observe any cell death in other wells with other ratio of DNA and lipofectamine.
For my first transfection, my cell density was about 95% confluence but later I thought that probably by reducing the cell density to about 70% would work better as my cells always tend to clump at a foci.
Hi,
are you sure that your protein is well transferred to the membrane (have you stained your gel with Coomassie after transferring or stained the membrane with Poncou?) Or what about your antibodies? Do you have postive control? Your problem might be at those stages too.
Cheers
are you sure that your protein is well transferred to the membrane (have you stained your gel with Coomassie after transferring or stained the membrane with Poncou?) Or what about your antibodies? Do you have postive control? Your problem might be at those stages too.
Cheers
Yes, I agree with you about this point. I am planning to repeat my Western blot by using PVDF membrane as I afraid that NC membrane may not bind well to my protein of interest.
Also, recently somebody in my lab prepared the Coomassie blue staining solution by using G250 instead of R250 and my seniors are suspecting that probably my proteins of interest were not stained properly as this two dye bind to different amino acid. Therefore, they are suggesting silver staining.
Anyway, thanks a lot. I will get back to my bench and repeat my experiments by taking your advices and suggestions into consideration.