Blue/white with pET15b, Rosetta2(DE3)?

oliver.starks from gmail.com via methods%40net.bio.net (by oliver.starks from gmail.com)
Tue Apr 21 09:26:50 EST 2009


I've got my gene inserted in pET15b, and I transformed Rosetta2(DE3).
Colonies appeared in appropriate numbers on an Amp/Cam plate,
indicating the cells must have the vector.  Without thinking it out I
re-streaked a couple colonies onto another plate with IPTG/Xgal to re-
test insert presence (I'm fairly confident in my original ligation but
it's always possible that the cells from which I prepped my plasmid
were cotransformed with empty pET15b as well).  These produced blue
coloration.  But after thinking about it more, it seems like either
blue should never be produced (regardless of insert presence/absence)
or should always be produced (regardless of insert presence/absence) -
because the insert is not disrupting a lacZ gene like it is in
pBluescript or something of that nature.

So...how's the blue being produced, and is it even evidence of my
insert not being present?

I'll be testing these with new preps and PCR or digest today or
tomorrow anyway but thanks in advance for your help.


More information about the Methods mailing list