importance of directionality in cloning - (May/25/2005 )
Hi everyone - I have been trying for months now to clone two independent genes into a single plasmid. Each piece has its own promoter, cDNA, and polyA tail sequence. However, the pieces have ligated in a "backwards" orientation - while the first piece is in its original 5' to 3' orientation, the second piece ligated into the plasmid in the opposite orientation. So piece #1 is "forwards" and piece #2 is "backwards." Am I correct in assuming that this should not matter, as long as the promoter and gene are in the correct orientation to each other?
Thanks for your help! - Jen
It should be OK provided that it does have its own promoter, KOZAK, and essential to have poly A tail sequence. The only worry is if you get run through the poly A tail then you could potentially get antisense transcripts forming, binding and reducing your mRNA level, so it does depend on the length of your tail.
I'd give it a go.