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Preparing Materials for RNAi

Preparing Materials for RNAi. 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.

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Preparing Materials for RNAi

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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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