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RNAi Preparation Protocol for NGM/AMP/IPTG

This protocol outlines the preparation of RNA interference (RNAi) plates for bacterial feeder strains. It involves melting NGM-Lite and labeling 20 small plates with plain NGM, followed by preparing NGM plates with AMP and IPTG. The use of these components is crucial for selecting bacteria containing plasmids with dsRNA for gene silencing (e.g., dpy-11, bli-1). Participants must follow the instructions to inoculate LB/AMP broth and seed NGM/AMP/IPTG plates with bacterial cultures, ensuring correct labeling and proper handling to facilitate successful RNAi experiments.

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RNAi Preparation Protocol for NGM/AMP/IPTG

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  1. Preparing Materials for RNAi

  2. 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

  3. 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

  4. 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)

  5. Next class period • Follow instructions on p. 9-10 #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

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