Crystals during X-gal staining

Paul N Hengen pnh at fcsparc6.ncifcrf.gov
Fri Apr 22 11:47:11 EST 1994


 In article <9404212135.AA05555 at mercury.med.pitt.edu>
 bsh at MED.PITT.EDU (Basavaraju Shankarappa) 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

> Sometime back there was an article in BioTechniques describing
> the use of DMSO as a solvent for X-gal instead of DMF.  I know that 
> DMSO is much more miscible with water than DMF.  I think that might
> solve your problem. 
>	Raj Shankarappa
> bsh at med.pitt.edu

Also, make sure you are NOT using a polycarbonate tube for any of the dilutions
or storage. If you do, the DMF will dissolve it until it hits the water. Then,
BLAM!...it will precipitate. You must use either glass or polypropylene.

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* Paul N. Hengen, Ph.D.                           /--------------------------/*
* National Cancer Institute                       |Internet: pnh at ncifcrf.gov |*
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