cloning/restriction analyses MAJOR PROBLEM - (Jan/20/2007 )
I have encountered a problem that is very frustrating and seemingly impossible. I am working with clontech's pIRES-DSred vector (see website if needed) and a gene of interest that is in TOPO clone. I am deleting a major portion of the clone: everything from the CMV promoter to the polyadenlyation signals because my cDNA has a poly A tail and I am using Ase I and Afl II enzymes to do so. I ligate my insert (1KB) and remaining pIRES vector 4kb. The transformation produces no colonies of vector only and quite a few on vector plus insert. My restriction analyses however makes absolutely no sense and to make matters worse the clone now seems to be resistant to ampicillin when it should only be resistant to kanamycin! My insert is a toxic gene that induces apoptosis in a eurkaryotic system if this sparks any ideas. I have repeated it several times with the same aberrant results. Is this even possible? You may want to check clontech's website to see what sequences I am deleting.
www.clontech.com
1.vector info
2.type in pires
3.click on piresdsred
www.clontech.com
1.vector info
2.type in pires
3.click on piresdsred
Hi,
The positive clones that you get should be a 5kb construction, which have a similar size with your Topo clone(4.9Kb). And the Topo clone just contains a Ampicillin resistance, so I suggest checking your Dsred Vector to make sure that it not be contaminated by your topo clone(or confused).
Hyland
But when I digest the pIRES-Dsred and topo, I am gel extracting the bands of correct size. If there was contamination, I would have suspect bands.