heterologous southern

Frauke Naumann fnaumann at scan.genetik.uni-koeln.de
Thu Mar 9 06:43:09 EST 2000



>I want to detect a beetle gene with a drosophila probe in a genomic
>southern. In one publication I read about a hyb-temperature of 37 degrees.
>Is that standard condition for such southerns? Does anybody have
>experience or refereces for such an experiment? (the probe is just about
>200 bp long)
>
>Thanks in advance
>
>Gregor


37°C should be hybridization in formamide (normally at 42°C). When I
hybridized a human cDNA to genomic DNAs of several species (mammals and
birds) I got signals even at high stringency (normal conditions, 68°C in
2xSSC-based hybridization solution). Depending on the conservation of the
probe you should try first stringent and then lower by 1-2 °C steps. When
you decrease the temperature too much you end up with unspecific signals
which won't help you anyway. Try also reduced washing stringency (like
higher salt, lower temperature)  - especially because your probe is quite
short.
I hope that helps
Frauke






More information about the Methods mailing list