I am planning a one-step gene disruption strategy, but have few
choices of restriction sites. In order to avoid fancier schemes, I
would like to use polylinker sites to generate the linear fragment
for transformation. However, I am concerned that the non-yeast bases
at the ends of the DNA fragment may interfere with
recombination/transformation.
Is my worry unfounded? Has anyone out there had good success with
gene disruptions using a linear piece of DNA that has 5' and 3'
"tails" of non-yeast DNA flanking the yeast sequences of interest?
How much extra sequence can you get away with in these cases and
still have good recombination at the site of interest?
Thanks in advance,
Robin Wright
