difficulty in cloning oligos into vector - cloning difficulties (May/19/2005 )
I have ligated a ~50bp DS oligo into a commerical vector (linearised with incompatible ends, Sal I and Xba I). The oligo was designed with a Sca I restriction site in the middle. After cloning into the Sal I site both the Sal I and Xho are lost. This allows quick screening of recombinant plasmids as plasmids with the insert are not linearised when digested with Sal I but are linearised when digest with Sca I (as this is only present in the inserted oligo).
However, when i performed the digests from three separate colonies, the plasmid was linearised with Sal I and Sca I which doesnt make sense cause digestion with Sal I is saying the insert isnt present but digestion with Sca I is saying the insert is present. Does anyone have any idea as to why i am getting these confusing results. Could it be possible that the electroporation resulted in the uptake of more than one plasmid i.e one with and without insert? Any suggestions how i could go about resolving this?
I am not sure if it is possible one cell can take up two or more plasmids. From my own cloning experience, it seems yes. At first, I thought the sequencing company had contaminated my samples or I had during plasmid prep. I repeated the plasmid prep and resequenced, I got the same results from some clones which show two different alleles. Another less likely possibility is the colonies I picked were actually merged from two colonies although I always only pick well isolated ones.