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Transient expression vector - (Aug/03/2009 )

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genehunter on Aug 13 2009, 01:38 AM said:

Although it is less defined than the approach of cloning into pBABE, it is possible. If you used a large ratio of the target vector (pCMVsport) vs the selection vector (pBABE), like 10 to 1, you have better odds. Plasmid integration is more or less a random event, if pBABE integrates, so should the pCMVsport. Unless your gene has negative effect on cell growth, it will stay.

You can select/verify target gene positive clonies from the stable clonies.

Usually i use 30ug of plasmid DNA for transfection but in a 1:1 ratio (15ug from each pBabe and pCMV).
Is this the problem why i'm not getting cells overexpressing the target protein?


Usually you will have different colonies that express at different levels. This is because they vary dramatically in chromosomal integration site and the copy number integrated. You need to do a limited dilution, get the colonies, and screen for individual colonie for protein expression levels.

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