Crystals during X-gal staining

Bernard Murray bernard at elsie.nci.nih.gov
Thu Apr 21 18:03:58 EST 1994


In article <CoMBtv.I1M at freenet.carleton.ca>, ar229 at FreeNet.Carleton.CA (Allison Haggarty) writes:
> 
> 
> I have been using X-gal to stain cells blue after transient tranfections. 
> The cells are blue allright but there are lots of crystals in the stain so
> the results are not very photogenic.  The problem seems to arise when the
> X-gal stock (10% in DMF, frozen at -20 C) is diluted 1:100 in the working
> buffer for staining (5mM K ferricynanide, 5mM K ferrocyanide, 2 mM MgCl2,
> made up in PBS).  As soon as the X-gal hits the aqueous buffer the
> solution goes cloudy. 
> Does anyone know how to prevent this? Am I stuck with filtering? 
> Thanks.
> 
> -- 
> Allison Haggarty

Hello,
	I use a slightly different protocol; 0.1 M phosphate pH 7.4 instead
of PBS, and I include Nonidet NP40 (0.02%) and deoxycholate (0.01%).
I add the X-gal (to 1 mg/ml final) as a 2% stock solution in DMF.

	When does the pptn occur?  If its just when you make it up then add
the X-gal solution more slowly (maybe use a more dilute stock solution).
If its when it hits the cells then make sure they have been rinsed well
after fixation.
	I routinely filter the staining solution (0.45 um filter) when I
make it at first and when stored in the dark it lasts for weeks.  When
precipitation does occur then it still works after another round of filtration.
	In the protocol I was originally given it was suggested that, after
a suitable level of staining had been achieved, the cells be washed with
phosphate buffer containing 3% DMSO.  I've never done this as I usually
photograph the cells first.
	I hope it all works out,
		Bernard


(What's wrong with filtering, anyway?)

Bernard Murray, Ph.D.
bernard at elsie.nci.nih.gov (National Cancer Institute, NIH, Bethesda MD, USA)




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