I am doing a cloning project where I'm inserting a 2kb gene fragment into 2 different plasmids; for one of the plasmids, the restriction sites are located such that the 5'->3' direction of the gene is in the same direction as the transcription direction arrows on the plasmid. But for the other plasmid, the gene would be going "opposite" the direction of the arrows. I was able to easily clone the gene into the "same direction" plasmid, but have yet to get any luck with the "opposite direction" plasmid, I was wondering if the directionality is why it's not going in?
PS--My boss is the one who designs the primers, I haven't learned how to do all that yet; I just work with what I'm given.
Thanks for any help, I've been registered here for a while, but this is my first time posting
Edited by rosephish, 26 September 2013 - 02:46 PM.