I am trying to create a stable cell line for a while now.
The purpose of the stable cell line is to express a protein with only half of a GFP protein, so that I could later transfect it with a protein holding the other half.
The problem is that I have no way of knowing if it's working or not during the selection period which can take up to a month.
I am working with HeLa cells. I did a death curve for G418 and use it according to the con. we have found. The cells are outgrowing the control plate (normal HeLa cells with G418), however after I transfect them with the other half of GFP and I don't see anything.
I read somewhere that I should cut the plasmid before transfection for stables. Is that correct? Perhaps, if I don't cut it, then when the cell would fuse the plasmid into the genome it will linease it at random locations - even in the middle of the gene of interest?
Normally you don't need to linearize the plasmid. It will be a random insertion and the chance of getting mis-integration is not that much high.
I would suggest you to insert your half GFP into another vector which has puromycin as selection antibiotic. It will be only 2-3 days selection then.
Second, is there any publication that uses your half GFP protein?
Are you sure that when two parts of the protein are expressed they are going to glow?
If you can explain your project litlle bit more detail, may be I can have more suggestions. You can use other methods...
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