xgal/iptg selection: summary

Paul N Hengen pnh at fcsparc6.ncifcrf.gov
Wed Jan 26 22:19:41 EST 1994


Recently, Craig Wilding wrote a review of messages about problems he was
having with the blue color indication of pBluescript...

> 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.

Within that compilation, I wrote:

> From: pnh at fcsparc6.ncifcrf.gov (Paul N Hengen)
>
> Caroline A Breitenberger writes:
>
>| ...To summarize, XL1Blue has an F' episome without which you cannot
>| do blue/white screening.  Check your cells on tetracycline to make
>| sure they retain the F'.
>
> A better control is to transform cells with uncut M13 DNA. If the E.coli has
> lost the F factor, you will not get plaques since M13 infects through the F
> pilus.  Selection of XL1Blue cells with tetracycline may lead to insertions of
> Tn7 in the chromosome because that is the source of antibiotic resistance on
. ^^^
> the F plasmid in that strain.

Oops! I really meant Tn10 (tetracycline resistance). Sorry for the mislead.
Also, I did some checking and found that F is not essential for infection by
the M13-like phages. Cells which lack F pili are more than 1000-fold less
susceptible to infection than those with F pili. So, essentially the lack of
plaques is still true when considering the turbidity within the top agarose.

: Eventually I pinned it down to a completely different matter. You will note
: that I was using Luria agar, naively assuming that any general purpose agar
: would suffice (I am a marine biologist not a bacteriologist/biochemist). This 
: contains an addition of sterile glucose solution. When I swapped to LB agar 
: which doesn't have this sucrose, the blue colour appeared, so I presume that 
: the bacteria were using this sucrose as an alternative sugar source and 
: staying well away from the Xgal. Perhaps.

Well, when glucose is present in the media, the Lac operon is repressed. Given
that you are using a vector with an operator site, this makes perfect sense.
But, the F plasmid of Xl1-blue contains a copy of LacIq anyway, so there is
plenty of repressor around. Adding IPTG is therefore necessary to express LacZ'
from vectors that are repressible. Now I'm really confused. pBluscript is
derived from pUC19 and therefore lacks the operator site (LacO). Addition of
IPTG is not needed with the pUC plasmids, only with the M13 cloning vectors
because they do contain LacO. So I don't know why you are adding IPTG to the
media in the first place if you are cloning in pBluescript phagemid vector.
If you were to grow M13mp19 on this strain, you would then need IPTG in order
to see blue plaques.

: I also found that my estimations of what 20ml of agar looked like in a petri
: dish were wrong and when I cut the volume down appropriately and thus got the
: Xgal/IPTG concs correct (I had been adding these to the plates after pouring 
: as I didn't need many plates) the blue colour was more intense- probably the 
: same colour as the sky above 12,000ft.

I would believe that the low amount or source of Xgal was the major player
here, and the media didn't make much difference. It's amazing how a simple
thing can sometimes hang you up :-)

-Paul.

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* Paul N. Hengen, Ph.D.                           /--------------------------/*
* National Cancer Institute                       |Internet: pnh at ncifcrf.gov |*
* Laboratory of Mathematical Biology              |   Phone: (301) 846-5581  |*
* Frederick Cancer Research and Development Center|     FAX: (301) 846-5598  |*
* Frederick, Maryland 21702-1201 USA              /--------------------------/*
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