Same insert twice in same vector?

Go to the profile of mchlbrmn
Feb 08, 2018
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How advisable is it to insert the same gene twice in the same plasmid vector, to try two different promoters? How stable or difficult to work with would that be?

2 Comments

Go to the profile of relaxin
relaxin 5 months ago

I have never tried this. But I think there will be no advantage in doing this. You can transfect cells with two different plasmids. Increased plasmid size will affect the yield and stability.

If you really want to do it, have the inserts being controlled by different promoters in opposite orientation.

Go to the profile of Roberto Rosati
Roberto Rosati 5 months ago

About stability, I think it might work. As you know there's a risk for recombination, and this risk would depend on the origin of replication / copy number, the strain used, and the extension of the duplicated gene; but, say, some high copy number dCas9 plasmids I recently acquired have two U6 promoter-driven sgRNA sites (about 360 bp each) and they're stable in DH5alpha cells.
I've also worked with BACs and PACs from the RPCI genome libraries, which are low-copy, and are also stable despite carrying 200kb inserts with several homologous human repeats in them.