pUC19 Blue/White riddle.
Keith.Rand at molsci.csiro.au
Sun Jun 20 21:15:36 EST 1999
In article <3767CC3F.AEC51F3A at purdue.edu>, Phillip San Miguel
<pmiguel at purdue.edu> wrote:
> I cut pUC19 with SmaI/XbaI, gel purify and ligate. I
> run on a gel and see 2 strong bands and a weak band at 2.7
> kb, 5.4 kb and 8.1 kb, respectively. I cut out and isolate
> the DNA from the 2.7 and 5.4 kb bands. I klenow fill (with
> all 4 dNTPs) and ligate. I transform into DH5alpha and
> plate on Xgal/IPTG ampicillin (actually methicillin, but you
> get the idea).
> Should the colonies be blue or white?
> My guess would have been "blue" for the 2.7 kb and "who
> knows" for the 5.4 kb. (The 5.4, I was guessing would be
> Xba/Xba (in inverted orientation) dimers mainly. I'm not
> clear on whether E. coli will replicate such a monster.)
Didn't you observe the 5.4 Kb band before you did any ligation? If so,
your pUC19 prep must have dimers or higher oligomers, and you only
achieved a partial digest. (You would probably then expect to see blue
colonies from the 5.4 Kb, which is not what you got, of course!)
Possibly your ligation didn't work and the only colonies that you got were
the result of transformation with linear DNA. In this case you might well
expect deletion mutants that would be white.
Keith Rand, Sydney Australia
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