xgal/iptg selection: summary

osp086 at vaxa.bangor.ac.uk osp086 at vaxa.bangor.ac.uk
Wed Jan 19 11:30:34 EST 1994


Well a considerable amount of time has elapsed since I posted the following 
request for help:

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.

The following are the summarised responses:

-----------------------------------------------------------------------------

What conc. are you using the XGal at? I suggest using at least 40 ug/mL
final conc., added to the molten agar, likewise for IPTG. Both added from
freshly made stock sol'ns ... XGal in DMF (example, 50 mg in 0.5 mL DMF,
added to 1 L LB agar ... IPTG in H2O, also made fresh, 50 mg in 1 mL H2O,
added to 1 L LB agar.

Also, 50 ug/mL Amp seems rather low ... I suggest increasing this to 200
ug/mL, since satellite colonies will surely form at lower Amp conc. Are
you seeing smaller colonies surrounding larger ones? Especially if plated
too densely (that is, more than 200 larger colonies per 10 cm dia plate,
will surely give rise to many satellite colonies (which will be white if
lac Z mutant strain is used).

Also, why do you incubate plates in fridge with hope of color development?
If it hasn't occurred after 24 hrs at 37C (once colonies are at least 1 mm
diameter), then it'll likely not occur at all.

Once again; are you adding XGal and IPTG from FRESHLY made stock sol'ns?
Weighing 50 mg per Litre isn't absolutely necessary either (especially if
one doesn't have mg weigh scale available) ... simply add small pinch at
end of spatula, into bottom of 1.5 mL microfuge tube, such that volume of
XGal powder takes up about 50 uL volume, then add 0.5 mL DMF, vortex, add
to molten agar at about 55*C, swirl immediately to ensure thorough mixing of
XGal. 

What volume of DMF are you adding to the agar? Note I add 50 mg XGal in no
more than 0.5 mL DMF per Litre agar. Much larger volume DMF may be harmful
to the bacteria.

best wishes,
                            ********************
Andre Hamel                              email: hamel at ccu.umanitoba.ca
Manitoba Veterinary Services          lab tel.: (204) 945-7630
545 University Crescent,                   FAX:(204) 945-8062 
Winnipeg, Manitoba, 
CANADA   R3T 5S6            ********************

-------------------------------------------------------------------------

From: Jun Li <jl74 at edu.columbia>
Dear Craig,
	I am suffering the same problem as you do. I have had some
successful selection before. But recently I got all negative. The 
solution in my case could have some to do with the transformation 
procedure and I am going to try out. Another point, concerning your
post, is that the IPTG concentration could be a factor because IPTG
can be a inhibitor of beta-gal if used in high concentration.
	Anyway, please give net a summary if you have a good solution
and I'll be glad to inform you if I solve my own problem.

Best wishes.

sincerely,
Jun Li

------------------------------------------------------------------------------

From: mbpfw at uk.ac.daresbury.seqnet

Don't focus too much on the X-gal. IPTG can and does go off! If it smells then I
think it should be considered suspect. It seems to be particularly necessary for
the Bluescript system and I have never had to use it for any other Lacz setup.
When we used our IPTG on Bluescript we got zip and this was corrected when we 
purchased some fresh IPTG. We had stored our old stuff for about a year at -20.

Worth a thought anyway

Phil

Phil Watson
Department of Medicine
University of Sheffield

----------------------------------------------------------------------------

Look at your bottle of Xgal, and make sure it is Xgal, not Xglu:i

	5-bromo-4-chloro-3-indolyl-B-D-galactopyranoside

and NOT 5-bromo-4-chloro-3-indolyl-B-D-glucopyranoside

cos the latter won't work!  

Cheers,

Martin

NNNN   NN  Martin A Kennedy (E-mail = mkennedy at chmeds.ac.nz)  ZZZZZZZ  
NN NN  NN       Cytogenetic and Molecular Oncology Unit          ZZZ
NN  NN NN           Christchurch School of Medicine            ZZZ
NN   NNNN              Christchurch, New Zealand              ZZZZZZZ
	      Phone (64-3)364-0880  Fax (64-3)364-0750
================== RFC 822 Headers ==================

---------------------------------------------------------------------------

From: brianf at med.uvm.edu (Brian Foley)

	Are you sure that the E coli XLI-Blue that you used still have the
F plasmid in them?  If so, they should be tetracycline resistant.  The 
F plasmid carries the other part of the beta-galactosidase that is needed
to combine with the part produced by the pBlueScript plasmid.  So it is
possible for the XL1-Blue to loose the F plasmid and thus give you all
white colonies.

	Next, try increasing the ammount of Ampicillin to 70 or 100 ug per
ml.  I find that some XL1-Blue can grow on 50ug/ml Amp without being
transformed to true ampicillin resistance.  But you should know if this
is happening by seeing as many colonies on a plate with cut (no ligase)
vector as on the plate transfromed with uncut vector.  So the symptoms of
white colonies from untransformed cells is different from the symptoms
from having lost the F plasmid.

	Next try a different batch of X-gal, or ask a friend for a sample
of transformed E.coli that he/she knows to produce a dark blue color on a
good X-gal plate.  It sounds like you already tried a different X-gal plate
by borrowing one from another lab.  

	I find that it sometimes take a while for a deep blue color to 
show up.  Let the colonies grow at least 14 hours at 37 degrees, and
then give them at least 4 hours at 4 degrees.  A colony of XL1-blue
containing both the F plasmid and pBlueScript should be a dark sky-blue
(darker than the sky ever looks except when you are above 12,000
feet in the mountains on a sunny, dry day and you have sunglasses on)
by the end of this time.

	Please let us know what the end result is.
--
********************************************************************
*  Brian Foley               *     If we knew what we were doing   *
*  Molecular Genetics Dept.  *     it wouldn't be called research  *
*  University of Vermont     *                                     *

--------------------------------------------------------------------------

From: "RITCHINGS, BRUCE W" <BWR at edu.ufl.ifas.gnv>

Your problem is easily solved. XL1Blue contains Tet resistance AND the other
half of the BGal gene (for the life of me I can't recall which half) on the 
F' episome. Ergo, if you don't add Tet to EVERY solution which XL1Blue 
touches you risk losing the episome, and with it, your color selection. 
Ergo, put 50 ug/ml Tet in your media, both when you're just growing the 
plasmid to make more, as well as to your plates when doing selection. By the
way, Tetracycline does not fully dissolve in either ethanol or water (I 
mention this because I routinely see people making it up incorrectly in our
building). I make a 15 mg/ml stock in 75% ethanol and it works great. 
Stratagene says not to go over 50 ug/ml, but I wouldn't go under either. 
Now if I can just remember how that alpha-complementation business works for
BGal! Cheers, Bruce Ritchings, Univ. of Florida

-----------------------------------------------------------------------------

From: reinhard at wst.edvz.sbg.ac.at (NESTELBACHER Reinhard)

I had the same problems (The biggest problem of mine is, that I can't wait)

1.) Try to let your plate longer on 37`C (if the size allows this).
    Maybe there is not enough beta-Galactosidase

2.) Check the pH of your plates. It should be not lower than 7.0
    The pH of my plates is about 7,5 adjusted with NaOH before (!) autoclaving.

3.) How to you transform the cells?
    Which protocoll for competent cells?
    Do you have to preincubate before spreading it on the plates?
    If yes, than give IPTG (it can be a lower concentration) to this medium
    You would not need it in the plates.

4.) Try to make Amp-plates without XGal but speat it on the plate before using it
    ( 100 microltr. of 20mg/ml)


Good luck! Please inform how you solved the problem!!!!!

NESTELBACHER Reinhard
reinhard at dgen10.sbg.ac.at
University of Salzburg/Austria
Institute of genetics

---------------------------------------------------------------------------

The above suggestions are all good ones. But another possibility is that 
your XL1blue cells have lost their F' episome and in this case they
will no longer turn blue when carrying a plasmid. Be sure your cells are
still Tet resistant.


----------------------------------------------------------------------
 Michael Benedik				INTERNET: Benedik at uh.edu
 Dept. of Biochemical & Biophysical Sciences	
 University of Houston				BITNET: Benedik at uhou
-----------------------------------------------------------------------

--------------------------------------------------------------------------

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. I've always wondered about this, so does anyone
know what makes F' more stable in DH5aF' than in the older JM101 or JM109
strains?

*******************************************************************************
* 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              /--------------------------/*
*******************************************************************************

Well I have at last solved the problem. I tried screening for tet. resistance
and it indeed gave me lower counts so this may have played a part in the 
problem but I don't now think it was the major contributor. I thought about
smelling the IPTG to see if it had gone off but considering all the toxic by
inhalation and general death warnings on the bottle I thought i'd give that a
miss: thanks Phil.
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.
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.
So there we have it. Thankyou all very much for your helpful suggestions and 
my apologies for taking so long to reply with a summary.




More information about the Methods mailing list