Need to sequence gel purified vectors exposed to UV? - (Jun/22/2008 )
I have some constructs that I have cloned which do not work as expected. I have sequenced the insert region of these plasmids to rule out mutations in the PCR product I used as the insert. However, the backbone also contains some important genes that are not related to antibiotic resistance.
The backbone fragment was gel purified by visualizing the bands on a UV transilluminator. I am very mindful of minimizing exposure of my DNA fragments to UV, and I was successful in obtaining ligation products that transformed into E. coli. I know UV induces mutations which drastically reduce transformation efficiency, but does anyone know how frequent UV-induced point mutations are in "transformable" ligation products in which the selectable marker is functional?
The backbone fragment was gel purified by visualizing the bands on a UV transilluminator. I am very mindful of minimizing exposure of my DNA fragments to UV, and I was successful in obtaining ligation products that transformed into E. coli. I know UV induces mutations which drastically reduce transformation efficiency, but does anyone know how frequent UV-induced point mutations are in "transformable" ligation products in which the selectable marker is functional?
I may be wrong, but UV induces double stranded breaks, and that drstically reduces the transformation effi.of those DNAs. Now, those breaks may be corrected once inside, and may result in some mutations, but not much.
It is always good to sequence the important genes in your vectors anyways, unless you have some selection strategy to check their functionality.
The backbone fragment was gel purified by visualizing the bands on a UV transilluminator. I am very mindful of minimizing exposure of my DNA fragments to UV, and I was successful in obtaining ligation products that transformed into E. coli. I know UV induces mutations which drastically reduce transformation efficiency, but does anyone know how frequent UV-induced point mutations are in "transformable" ligation products in which the selectable marker is functional?
I may be wrong, but UV induces double stranded breaks, and that drstically reduces the transformation effi.of those DNAs. Now, those breaks may be corrected once inside, and may result in some mutations, but not much.
It is always good to sequence the important genes in your vectors anyways, unless you have some selection strategy to check their functionality.
Thanks cellcounter. I was hesitent to sequence the entire region of interest becase it spans almost 7 kb, but I will make this a priority if I can't soon verify functionality of the important genes (which basically allow for in vivo viral amplification of the gene of interest on the same transcipt).