Transformation using linearized plasmid? - (Apr/14/2006 )
I recently cut out a portion of a vector. This vector contains genes resistant to Kanamycin. If this vector is not religated, can it still provide kanamycin to cells after transformation? (note the kanamycin genes were not cut out). Does a vector have to be circular in order for transcription to occur?
A linear fragment will not confer resistence as a plasmid. In some strains you can use linear fragments to insert or replace existing genomic or plasmid sequences to insert markers such as Kanamyacin.
plasmids have an origin of replication that ensures the replication in both senses. But at the ori site, there is a tough process for the new synthetised molecule to be ligated (and so ciular). Assuming your vector is cutted, it can't be circular. So it can't replicate. So there is no kana resistance.
But if you have done transfowith a cutted vector, it always remains some undigested/simple digested vector that can give colonies
Thanks a lot guys! you guys hit the nail on the head for me!!!!!!
Linear plasmids will transform cells because they can be re-circularised (remember that E.coli has its own DNA ligase). As a general rule of thumb assume that linear DNA transforms at 1/100th the efficiency of circular DNA.
DNA sequencing services
Agree with Daniel, if I'm not mistaken, before ligases were available that's the way transformation to create insertions or deletions were done: transform cut vector + PCR product (or part of a cut vector). Somehow it worked (though not really efficient).
Many years ago I linearized a plasmid with HinD III and lightly digested the ends with Bal 31. The plasmid was ligated and then recut with HinD III to linearize anything that was not digested with Bal 31. Transformation into C600 yielded hundreds of colonies, which were all religated HinD III-cut plasmids. After treating the ligated and HinD III-digested DNA with CIAP, the high background went away and I got the desired products.
So, it is possible to transform linearized DNA. Are there strain differences in regards to transformation efficiencies(EndA plus/minus and other factors)? Are there differences between restriction fragment sequences?