our cell line will be transfected with 2 different plasmids at the same time for 48 hours but the results is not equal between all wells and the results should show us an equality so my question is:
Is the cell culture a zero order or a first order kinetics and how can we adjust molecular growth between viable cells to show the same rate of COD transfection equalization?
for example, should I make several gradient concentration from a plasmid A with several gradient from plasmid B and finally mix all of them to equalize the same amounts of transfected plasmids at all cells?
Hi Ahmed, do you prepare the master transfection mix or do you prepare mixes separately for each well? You can increase exponentially the chances of obtaining the equal transfection efficiency by first preparing the master mix and dispensing it in the individual wells. If you are using a multi-well plate, I would suggest using only the middle wells, and filling the side wells with water or PBS, because evaporation of medium from the side wells can also sometimes result in bizarre outcomes.
I'm making different wells, but your answer about using middle wells will be a technical useful.
and I decided to make 3 different concentrations of my transfection for all mutants and mix all of them for each separated well together then I will show the best matching equalization.
if you have a better solution I will be grateful if you share knowledge
I guess, I misunderstood your original post. So every well in the plate is getting a combination of two different plasmids, right? If so, this precludes the possibility of making master mix.
I agree with your experimental plan. It seems that you will have to empirically determine the right concentrations of plasmids to obtain the equal expression levels in all wells. Do your plasmids express the point mutants of the same gene? If this is the case, and if you are using the monoclonal antibodies, the difference in the intensity of western blot bands could be due to the differences in antibody binding. You may need to use a different antibody if the mutations are in the antibody-binding region of your gene.
Good luck!
- Badges
- Science topic