pcr product transfection - (Jun/20/2006 )
any ideas regarding direct transfection of PCR product into animal cells?
Naked DNA transfection is supposed to be inefficient. so it should be the same for PCR products unless u wish to use some reagents to transfect into cells.
What do you want to do? Use transfection reagents/electroporation to get PCR-product into cell lines? If so: what's the purpose?
It's possible to get PCR-product into cell lines, I do it for homogous recombination with vectors deleted for a certain region, and this works nicely (so both vector and PCR product have entered the cells).
Please shed some light on your experiments.
i am interested in transfecting a gene into a mutant cho cell line. and as i need to manipulate this expression system with a lot of inserts from the mammalian genome, i thought cloning it into a bacterial vector and transfecting it into the cell line would be laborious. instead if i can put them all into the vector, amplify the region of interest, along with the insert and directly transfect it, it would really save time for me. i heard it is of course tough, but then it is possible.
iam planning to do a calcium phosphate mediated transfection. we also have an electroporator. but i think i need to standardize protocol for it.
thanx in advance
So you would hope your insert would get into your vector by homologous recombination?
It's possible, as I do it. I use standard calcium phosphate techniques on 293T's, but it's possible on other cell lines as well (also with different transfection reagents/techniques.
You will have to check if your insert is actually there of course, and also probably need to select for stable clones with the insert, but it's possible.
Just make sure that there's a unique restriction site one the spot where you want to have your insert, cut your vector with this restrictin enzyme, precipitate your DNA together with your insert and transfect. That's the way I do it with rather great succes!
i will be specifically inserting the sequences into a unique EcoRI site in the vector. then i will amplify my region of interest which will include the insert(this is going to be an expression system). i then have to transfect this into the cells. my vector is 8.4 kb . my region of interest will be nearly 4 kb. so i will have to study the expression patterns of the gene after insertion.
If you ligate it into the EcoRI site, why don't you just grow the plasmid and transfect? I transfect plasmids up to 13 kb + about 3 kb of PCR product without any problem, so I believe your transfection wouldn't be so difficult
If you just transfect your pcr product, how are you selecting for cells being transfected? Not every cell will be transfected with your PCR product, so it might be a better idea to transfect your entire vector, and select for stably transfected cells and then study expression patterns. This is the harder way, but seems more suitable to me.
but the number of individual inserts which i will have will be toooo many. so i believe doing this way will minimize the tedious job of selecting them via a bacterial system.
yes. exactly. i ligate a mixture of DNA into a vector and PCR amplify an expression gene which will take care of the regulatory regions as my inserts are going to be present within the intron of this gene. my aim is to study whether these inserts have any influence on the gene expression.