While working with cell lines e.g HEK cell, CHO cell lines that I used mostly, what are the precautions for best transfection results?
Hello Zahir, for transfection, one of the most important things is the purity of the DNA and a correct proportion between liposome/DNA. For the first I recommend to use a good purification kit for trasnfection purposes (I like Qiagen). For the second problem you have make a curve to find right proportion for your specific line. Careful, the eficiency of the trasnfection also depends of the cell line, so try different curves for CHO and HEK, for CHO cells is so easy to trasnfect, for HEK I do not have experience. Best!, Adrian
An important thing I will recommend you is that do not pipette the Liposome and DNA complex vigorously, it will start getting the precipitate that will reduce your transfection rate for sure.
There are a number of variables some already discussed:
1. Good quality DNA: supercoiled is good for general tfx using liposome based reagents. Others use linearised DNA for electroporation.
2. Cells obviously need to be healthy and growing well. low passage number is good. CHO/Hek are both very easy to transfect.
3. The choice of reagent is very much up to you and what you want to achieve. For small scale transfections/routine testing we use Mirus (we have also used lipofectamine). For larger scale transfections we used PEI (very cheap small chain fatty acid)
4. Test ratios of DNA:reagent (also cell number). Use either a marker such as GFP or something you can quantitate in an assay easily (Elisa). Transfection effciency may well vary with size of plasmid or amount and cell number used. Also whether cells are adherent or in suspension.
5. Commercial reagents usually come with guidelines on certain media to use for transfection i.e. Optimem etc. So do check and they may give guidance on the transfection method: the order in which you add reagents may be critical
6. Do you need to select for transformants or are you just using a pool of transfectants. If selecting then your plasmid needs an selective marker present
Hi, apart from above comments,
Both CHO and HEK are easy to transfect cell lines. While performing different ratios, better use GFP having vector. It will be easy to find transfection ratio(GFP:Non GFP cells in visible are by fluorescent microscope). And it should be noted that the transfection efficacy varies by plasmid size, we have to use control vector(with out insert) in same quantity measured by agarose gel is preferable.
Hi,
I agree with all the comments. For these cell lines, it is easy to get > 50% transfection efficiency. If your goal is close to 90-100%, then you need to optimize all the conditions. Otherwise, your DNA quality is the most influence factor.
To select stable line, linear DNA may help based on many publications, but I did not feel that is important.
Yi
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