From owner-drosophila@net.bio.net Tue May 25 23:00:00 1993 Path: biosci!uwm.edu!ux1.cso.uiuc.edu!news.cso.uiuc.edu!blackmanr2.life.uiuc.edu!user From: Chris_Merli@qms1.life.uiuc.edu (Chris Merli) Newsgroups: bionet.drosophila Subject: Germline transform by electroporation Message-ID: Date: 26 May 93 17:45:11 GMT Sender: usenet@news.cso.uiuc.edu (Net Noise owner) Followup-To: bionet.drosophila Organization: university of illinois Lines: 13 I am attempting to do germline transformations of D. melanagaster using electroporation. I have been following the basic protocol of Kim Kamdar [NAR 20:(13) pp352; pers. comm.] but I noticed low survivability in non-dechorinated embryos at 250v/cm and 960uf. In short I collect 100-1000 embryos, 0-1 hr old, suspend them in 800ul of 10ug/ml plasmid + 2ug/ml transposase plasmid + 5mm KCl + 0.1mm sodium phosphate pH 7.8 at room temperature. I then pulse twice in a Bio-Rad electroporation apparatus, wait two minutes, drain, and place in fly bottles. I get less than 20% survivers under these conditions but I have not yet finished the crosses to determine the number of transformants. I would like to know if anyone else is attempting this method and what success they are having. Chris Merli .