Lactose RNAi induction

Eric J. Lambie Eric.J.Lambie at Dartmouth.EDU
Fri Apr 25 12:56:02 EST 2003


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


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