In article <3patbp$8en at nntp.ucs.ubc.ca>, mottus at bcu.ubc.ca (Randy Mottus) wrote:
> We have the CyO balancer with LacZ driven by the hunchback promoter and
> to do PCR on the non-balancer homozygotes which are lethal but will survive
> until late embryo. Thus I need a very reliable method for X-gal staining
> which does not make the chromosomes unusable for PCR. I'm told there is
> a very simple method but I haven't been able to find it as yet. Obviously
> it must be reliable due to the contamination potential for PCR. Thanks it
> advance for your help.
>> Randy Mottus
I have developed a method to stain unfixed, dechorionated embryos with
X-gal in 10 minutes and do western on them, I suppose it will work for PCR
The method is outlined in Rastelli et al., 1993, EMBO J. 12,1513-1522.
Pratically, after a standard dechorionation, one rinses the embryo in
triton, rinses again with PBS (NB need to elimintate the Triton, if not it
will mess up the interphase), treats with half volume heptane/half volume
PBS (this is to make holes for the X-gal to go in) for 10 minutes,
eliminate the heptane, rinses in PBS, rinses in Xgal buffer without the Fe
salt, put the embryo in depression dish, add X-gal staining mix, mix well
and put at 37 for 5-10 minutes and they stain.
Let me know if you need any further help
Dr. Luca Rastelli
HHMI/Baylor College of Medicine
Email: rastelli at bcm.tmc.edu