Replacing puramycin in a pLKO1.puro - (Oct/18/2012 )
Hi I want to replace the PuroR cassette with dTomato. I don't have a lot of experience with this and was wondering how I tell the limits of the size of the fluorescent tag that will fit in a pLKO1.puro? I think dTomato is like 690bp while Puro is like 599? What else should I take into consideration? Ive heard inserting tdTomato won't work in my system because of the dimer conformation, not sure why. Another question I have relates to the hPGK promoter that is driving puromycin expression in the pLKO1.puro plasmid vector, does this have any effect on dTomato after sub-cloning, does it have to be excised and replaced as well to effectively drive expression of dTomato? Any thoughts or suggestions on how to most easily do this are really appreciated. Thanks!
The size of dTomato should not be a problem. Don't mess with the promoter that is driving the expression as you will need that for good expression. As for how to go about doing this, find two restriction sites that can excise the PuroR cassette and amplify the dTomato by PCR and engineer these restriction sites into dTomato. Then you ligate the insert into the vector and you are good to go.
One thing to take into consideration is, once you remove the PuroR cassette you have lost a selectable marker. Unless this plasmid has another marker you have no way of selecting for it and this will cause problems.