ligation--pls help! - (Jan/05/2011 )

Hi Janani.
While trying to ligate into a 3.5 kb vector, I experienced 7-8kb bands when linearizing the DNA from minipreps, which corresponded to a 4-5 kb insert - not what I expected to clone Sequencing revealed that I had managed to clone a gene from a Pseudomonas bacterium which is commonly found in water! When I checked the sequence of the pseudomonas gene it contained an EcoRI and a NotI restriction site which perfectly complemented my digested vector. Quite an eye-opener!

Moral of the story: use a good source of water and don't run agarose gels in a bath that's been sitting there for the last 12 months