I use lipofectamine 2000 reagent for transfection. I am using 6 well plates for the experiment. So, I have these two plasmids which I need to transfect say X and Y. What I generally do is :
1. Take 4ug of plasmid X and add 250ul of optmemem (label tube as 1)
2. Take 4 ug of plasmid Y and add 250ul of optmem (label tube as 2)
3. Take 500ul of optmem and add 20ul of lipofectamine (label tube as 3)
4. Incubate 1,2 and 3 for 5 minutes
5. After 5 minutes, take 250ul from tube 3 and add in tube 1 and also in tube 2 respectively
6. Incubate for 20 minutes
7. Now add tube 1 containing plasmid X + lipofectamine (500ul) to the well 1 of the lablled wells in the 6 well plate and also add tube 2 containing plamsid Y + lipofectamine (500ul) to the well 1 for cotransfection
My questions are:
1. Do you think the protocol is ok?
2. Do I need to combine the plasmids+ lipofectamine for cotransfection before I add them onto the wells? If yes, then at which step?
never worked with lipofectamine.
We use calcium-phosphate transfection. Mix both of plasmids, rest of the components as it is supposed to be by the protocol and transfect.
What, do you think your plasmids will fight if to mix them? As far as I understood, your goal is to achieve the highest possible rate of co-transfection? So, then why to make the things even more complicated? What do you think - what are the chances that the same cell will get those 2 different liposomes? I guess it will be necessary to ask some mathematitias to calculate the probabilities...
You can combine both plasmids, in first step u can take plasmid x and y add together in optimum mix well andlabel tube 1 incubate for 5 mints, then u can add lipofectamine+ optimum tube 2 to tube 1 and incubate for 20 to 30 mints and then mix in each well of sie well plate
We transfect upto 8 different plasmids using this system for Influenza virus reverse genetics. We just mix them all together in one tube and they work perfectly every time. With a high transfection efficiency of around 80-85%, increasing the number of plasmids would only bring down the probability of getting all of them into one cell down to maybe 25-30%. In your case just 2 plasmids won't affect this probability much at all.
Thanks Thomas Ciucci, Mahadev Malhi, Yaroslav Tsytsyura and Sayantan Bose for your suggestion. Thank you very much for clearing my doubts.
Mix the 2 plasmidstogether before adding Optimem/ serum-free media (co-transfection upto 4/5 plasmids is very common) and follow the protocol for Lipo2000...should work just fine....to increase the transfection efficiency, you can keep your cells for 40-48 hrs (provided that suits your expt. scheme).....
If we want to do cotransfection with multiple plasmids. What would be the Ideal ratio ?
I have a similar question. I need to cotransfect two plasmids: CFP conjugated with protein of interest and YFP also conjugated with protein of interest. According to the manufacturer's protocol for 24well plate I should take 0,5ug of DNA and 2ul of lipofectamine. Do I understand corretly that I should take 0,25ug of my CFP_Protein plasmid DNA and 0,25ug of YFP_Protein plasmid and use it with 2ul of lipofectamine? Or perhaps when i want to contransfect i ought to use 0,5ug of EACH plasmid and 2ul of lipofectamine?
so finally can anybody conclude, what the final protocol for co transfecting two plasmid vectors is if one is carrying an insert to be expressed and other carrying an oligo to downregulate it?
For transfecting multiple plasmids, if they have some kind of correlated effects then you should use the correct molar ratios as well. Granted, larger plasmids will have lower transfection efficiency than smaller ones, corresponding molar ratios are easy to explain when publishing research. Cotransfection can be improved with reagents specific to your cell line (https://altogen.com/products-index/) and the use of selection markers if you need a more homogenous population.
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