Transient expression vector - (Aug/03/2009 )
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.
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?
-chijing-
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.
-genehunter-