PREP of NGM/AMP/IPTG I have melted down NGM-Lite One volunteer- please label 20 small plates – NGM only Pour 20 plates of plain NGM With remaining NGM, I will add Cholesterol, IPTG and AMP You will label plates NGM/AMP/IPTG Pour 20 as a class

Prep of RNAi Bacterial Feeder strains You will follow instructions on p. 8-9, #4-8 to innoculate some LB/AMP broth with your feeder strains Make sure it is LB/AMP – I will put the amp in for you

Why are we doing this? We are making special NGM agar The plates have amp TO SELECT FOR BACTERIA WITH PLASMIDS IN THEM THE PLASMID HAS THE dsRNA TO TRANSCRIBE The plates have IPTG We’ll put feeder bacteria and IPTG in the plate will ACTIVATE THE T7 PROMOTER, INITIATING TRANSCRIPTION OF THE dsRNA FOR THE GENES WE ARE SILENCING (dpy-11, bli-1)

Next class period Follow instructions on p #3-6 to seed your NGM/AMP/IPTG plates with RNAi feeder strains Label plates with which strain you are adding Add 100 microliters of bacterial culture After broth soaks in, invert, parafilm and store on benchtop