From news.indiana.edu!vixen.cso.uiuc.edu!news.uchicago.edu!newsfeed.stanford.edu!bloom-beacon.mit.edu!hgmp.mrc.ac.uk!not-for-mail Thu Apr 26 14:09:28 2003 From: Eric.J.Lambie@Dartmouth.EDU (Eric J. Lambie) Newsgroups: bionet.celegans Subject: Lactose RNAi induction Date: 26 Apr 2003 14:09:28 +0100 Organization: BIOSCI/MRC Human Genome Mapping Project Resource Centre Lines: 30 Sender: nobody@hgmp.mrc.ac.uk Approved: wmorgan@acs.wooster.edu Message-ID: NNTP-Posting-Host: mercury.hgmp.mrc.ac.uk X-Trace: niobium.hgmp.mrc.ac.uk 1051362568 28672 193.62.192.80 (26 Apr 2003 13:09:28 GMT) X-Complaints-To: news@net.bio.net NNTP-Posting-Date: Sat, 26 Apr 2003 13:09:28 +0000 (UTC) Xref: news.indiana.edu bionet.celegans:84 In my lab we've been using the Fraser et al. & Ahringer RNAi feeding library to screen for suppressors of gon-2. Since the smell of the IPTG was causing headaches among some of the lab members, we decided to try using lactose instead. In trial experiments, 0.2% lactose has outperformed IPTG, both in terms of efficiency of mutant phenocopy and in terms of overall lawn quality. We haven't tried huge numbers of different constructs or a wide range of conditions (1% lactose didn't seem any better), but it looks very good so I thought I'd let people know that this is a reasonable thing to try. Let me know if it works (or doesn't work) for you. The media we've been using is NGM-lite, supplemented after autoclaving with CaCl2 (to 1 millimolar), tetracycline (to 12.5 microgams/ml) ampicillin (to 100 micrograms/ml) and lactose (to 0.2%; added from a 20% filter-sterilized stock). NGM-lite mix (for 40 liters worth of powder): 60 grams NaCl 160 grams Bacto Tryptone 120 grams KH2PO4 20 grams K2HPO4 320 milligrams cholesterol 800 grams agar Mix well and use 29 grams per liter. Happy screening, Eric Lambie --- .