mysterious 1.6kb band after transformation - (Apr/25/2005 )
I am trying to clone a 1.8kb insert into a ~8kb vector. My pcr products/digests look fine. This is a cohesive/sticky-end ligation. I use electroporation to transform my cells (DH5alpha) and plate on ampicillinLB plates. After the transforamtion, I get colonies. However, after the miniprep and digests (EcoRI and KpnI) I get a mysterious band at 1.65 kb consistently in evey clone. I see no vector band and no insert band. Others in my lab who have successfully cloned their constructs remember seeing this mysterious band as well but no one knows what it is or how to avoid it. Has anyone ever seen this band and know how to get rid of it?? Any advice would be much appreciated!!
i encounter a dna contaminetion in my lab to.
To get rid of i change ligase and ligase buffer, reprepare new competent bacterias from a stock of an other team and by the way all solutions to make competent bacterias, and trated electroporation cuves with NaOH / sonication / sterilization.