I am using HEK-293T cells for transfection with plasmid DNA of pNL4-3 and p-INDIEC-1. I am getting good expression of pNL4-3 using Lipofectamine 2000 and cells are also in good condition after taking supernatant of 24 hours.
But in case of p-INDIEC-1, the expression is not good and even the HEK cells became nonadherent also. I am using 2ug DNA and preparing the sample according to Lipofectamine 2000, invitrogen.
What will be the ways to encounter this problem?
Hi Bivek,
Depending on the plasmid (length, composition in bases, method of purification, salts contained in the buffer...) transfection efficiency can vary from one to another.
Moreover, the promoter driving gene expression can also play a major role in the transfection efficiency:
- For instance, transfecting wih 2µg of a 4.5 kb plasmid will give a better efficiency than transfecting a 10 kb plasmid.
- buffer composition and method of purification will play a role also in transfection efficiency more than the 260/280 ratio
- plasmid "quality" (coiled, or supercoiled) is a key parameter
- promoter strength is a real important parameter that can induce several fold of efficiency in transfection.
one option in order to raise the transfection efficiency would be to try an alternative to L2000. You could try DreamFect reagent that have shown real good results in HEK cells (as assessed by may publications of whom :Murfuni I. et al, (PLoS Genet. 2013 Oct;9(10):e1003910), Fuchs Y. et al., (Dev Cell. 2012 Sep 11;23(3):611-23) or Lopez-Meija IC. et al, (Hum Mol Genet. 2011 Dec 1;20(23):4540-55.)).
This reagent presents a real high level of nucleic acid compaction (so that even long plasmid can be easily transfected), and moreover is biodegardable.
Please tell me if you need more information,
all the best,
Cedric
Thank you Cedric for useful info.
I was using nearly same size of plasmid, pNL4-3, the transfection efficiency and viral production was good for that, but not with of pIndie-C1. I have transformed plasmid DNA in TOP10 competent cell using heat shock, cacl2 method and then preparation and isolated the plasmid using genJET maxiprep kit. I got the fair quality band in gel. The plasmid DNA are kept in elution buffer.
I want to know about the proper method of transformation of pIndie-C1 and their transfection methods.
Thank you
Regards,
Bivek
Thank you Barbara.. But that much of HEK cells would be much more if we incubate for overnight, say 18 hours. I used to inoculate 5 x 10^5 cells per well in 6 well plate. As the HEK cell divide faster than the other.. and the size of pIndie-C1 plasmid is large. this cause hinder.
Regards,
Bivek
Hi Bivek
Did you see this paper
http://online.liebertpub.com/doi/pdf/10.1089/088922299310223
Not very recent but they talk about pIndie-C1 transfection.
if you can, maybe you coud try other cell lines
Nicolas
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