Colour selection problem on XGal/IPTG plates

osp086 at vaxa.bangor.ac.uk osp086 at vaxa.bangor.ac.uk
Thu Dec 2 07:08:34 EST 1993


I am trying to clone some fragments of mitochondrial DNA into bluescript
but am having trouble with the subsequent colour selection of recombinant
clones. I have cut the bluescript psk and the mtDNA with EcoRI and HindIII
and treated the bluescript with alkaline phosphatase. Ligations were
performed using the protocol of Maniatis et al. except for an excess of 
DNA ligase. The products as well as uncut bluescript, bluescript cut +
ligase and bluescript + no ligase (controls) were used to transform fresh
competant E.coli XL1Blue. These were then plated on Luria agar plates
containing ampicillin (50ug/ml) and XGal and IPTG at the levels stated in
Maniatis. After growth for 24 hours the plates were transferred to a fridge
to allow colour development. This however did not occur. The bacteria seem
to grow at the correct levels compared to the controls but don't change 
colour to allow selection. Sometimes some of the controls have adopted
a debatable blue-green tinge but nothing like the white and blue colours
I expected. I transferred some of the colonies to an agar plate produced
in another department but these didn't change colour either. I have also
tried spreading the XGal/IPTG as well as incorporating it into the 
medium.
	Can anyone suggest an explanation or cure?
Thanks in advance,
		Craig Wilding.

		School Of Ocean Sciences,
		University of Wales (Bangor),
		Menai Bridge,
		Gwynedd,
		LL59 5EY,
		Wales, U.K.



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