I have been cloning TOPO vector containing 3'UTR of my gene of interest into pmirglo vector (7350 bp). I digested pmirglo and topo vector with XhoI and SacI and ligated them. I also digested ligation reaction with NheI to digest self ligated pmirglo vector. After transformation, I obtained clones and isolated their plasmid. Then, I digested plasmid DNA and when I analyzed them on agarose gel their size are even shorter than original vector. I think it may be resulted from the ligation reaction but I repeated them several times. Has anyone encountered this problem, if so can you explain your solution?
Maybe you should check if your gene of interest has multiple cutting sites for Xhol and Sacl.
I digested TOPO vector with XhoI and SacI, then I isolated my insert from agarose gel before ligation reaction. Thanks for your answer Vikki.
Hi Ece, so you gel extracted your insert before the ligation? Did you do the same with both plasmids?
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