From owner-drosophila@net.bio.net Tue May 16 23:00:00 1995 Path: biosci!bcm!NewsWatcher!user From: rastelli@bcm.tmc.edu (luca rastelli) Newsgroups: bionet.drosophila Subject: Re: X-gal Staining Live Embryos Date: 17 May 1995 18:32:58 GMT Organization: Baylor College of Medicine Lines: 31 Message-ID: References: <3patbp$8en@nntp.ucs.ubc.ca> NNTP-Posting-Host: 128.249.46.114 In article <3patbp$8en@nntp.ucs.ubc.ca>, mottus@bcu.ubc.ca (Randy Mottus) wrote: > We have the CyO balancer with LacZ driven by the hunchback promoter and I want > 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@bcm.tmc.edu .