I am using BP clonase II and LR clonase II. entry vector is pENTR221. and Destination vector is pET-DEST55. I have 4 genes to clone and none of them are working. I have sequenced entry clones of all 4 genes in pENTR vector.
After performing LR cloning, I get colonies (TOP10 cells), I grow them in 2ml LB with carbenacillin, extract the plasmids and transform them into BL21 cells, I dont get any colonies there. BL21 cells are correct and competent enough, i have checked. plates are the same and working properly. other all things are proper i suppose. but still, somehow i am getting false positives after LR cloning step. I dont get any plasmid after growing cell in TOP10.
What could be the problem ? Help me oh Master!!!!