pUC19 Blue/White riddle.

Phillip San Miguel pmiguel at purdue.edu
Wed Jun 16 11:09:35 EST 1999


    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.)  I
picked "blue" for the 2.7 kb band because SmaI/XbaI+Klenow
fill-in should result in a 9 bp deletion from the pUC19
polylinker.  I would expect this to leave lacZ in frame.

    My result was white colonies in both cases.  Should I
suspect my Xgal/IPTG has gone bad?  (I think the antibiotic
was okay, I didn't get *that* many colonies.)

Phillip San Miguel
Purdue Genomics Center




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