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||What size of plasmid can be transfected?||A1||Plasmid DNA up to 200-250 kb can be transfetcted inside cells using GeneCellin and GeneCellin HTC. However, the transfection efficiency may decrease with the size of the plasmid.|
||What kit should I use to purify my DNA?||A2||DNA quality is very important for efficiency. We recommend using Qiagen mini-prep or endotoxin free purification kit|
||Can I perform co-trasnfections (2 DNA plasmids)?||A3||Yes. Both GeneCellin and GeneCellin HTC work for co-transfections.|
||When I used GeneCellin the transfection efficiency of my cells improved, but the cell viability dropped. Is there anything I can do to reduce the cytotoxic effect?||A4||Yes, it is best to titrate down the amount of DNA while keeping the same amount of GeneCellin. The cytotoxic effect observed is usually caused by the DNA. The amount of DNA which enters into the cells will increase with efficient transfection reagents resulting in toxicity. This is especially true if the quality of DNA is not very good or if the cells are sensitive to transfection.|
||I want to perform a transfection with more than one DNA plasmid at the same time.||A5|| |
There are three things to consider for multi-plasmid transfections:
i) The total amount of DNA used must be the amount indicated in user manual. For an example with a 24-well plate, we indicate to use 0.5 µg of DNA with 2 µL of GeneCellin. If you want to transfect 2 plasmids, then please use 0.25 µg of each (0.5 µg total amount) with 2 µL of GeneCellin (0.125 µg of each plasmid for 4 plasmids co-transfection....). If protein expression is low due to the plasmid dilution it is possible to increase the total amount of DNA. We do not recommend to use more than 1 µg of DNA per well in a 24-well plate.
ii) The plasmid size is also an important parameter to maintain equivalent expression levels. If we keep the example above and you co-transfect two plasmids (6 kb and 12 kb), then If you use 0.25 µg of each, you will have twice the number of molecules for the 6 kb plasmid. In this case it is recommended to increase the DNA amount of the larger plasmid (0.33 µg of the 12 kb plasmid and 0.16 µg of the 6 kb plasmid for a total of 0.5 µg of DNA).
iii) If your plasmids have the same promoter there is no problem. However if the promoters are different and one is stronger than the other, you will have to optimize the amount of DNA for each plasmids to obtain approximately the same level of transgene expression. In this case there is no other way than to empirically optimize conditions.