Insert Positive blue col

Thu Jan 21 15:29:00 EST 1993

We have also seen that in many clonings into the pUC 
lacZ MCS we end up with quite a few yeast DNA
fragments that give various shades of blue.  
If we get just blue  (no shade differential)
and white colonies the blues are usually non inserted.  But if we see 
one or two different shades of Blue clonies, including whites,
 on a plate it appears that the whites represent one orientation 
of the insert in the MCS and the lighter blue represents the 
other orientation of the fragment in the MCS.  On occasion I have
run across some fragments (1.1 kb URA3 Hind-Sma) that  are as blue 
the non-inserted vector in one orientation (blunt end ligation) and white
in the opposite ori.  One way we can sort of tell the difference between
inserted and non-inserted blues with  pUC plasmids grown in 
DH5 alpha is that the non-inserted blues do no grow very well giving 
very small colonies, were as the inserted blues tend
to be bigger healther looking colonies.  This works most of the time
but not always.  It's insert (DNA fragment) dependant.

Dan Gietz
U of Manitoba

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