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BISC219/F11: RNAi Lab 9: Difference between revisions
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→Lab 9: Series 3 Reverse Genetics-Induction of HT115(DE3) feeding strains for RNAi
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#Remove 3.5 ml of the supernatant. | #Remove 3.5 ml of the supernatant. | ||
#Resuspend the bacterial pellet in the remaining 1.0 ml of supernatant - you are concentrating your bacteria. | #Resuspend the bacterial pellet in the remaining 1.0 ml of supernatant - you are concentrating your bacteria. | ||
# | #In the laminar flow hood in L304 (the 220 lab) pipet a 200 μL aliquot of your induced bacteria onto the center of 4 '''feeding plates'''. Be careful not to tilt or jostle your plates so that the bacteria stay in a circle in the center of each plate. These plates contain the same NGM Lite medium used in our mapping series, except that they have been supplemented with 0.4 mM IPTG, 50 μg/mL ampicillin. | ||
#Allow the bacteria to be absorbed into the media before you move them. | #Allow the bacteria to be absorbed into the media before you move them. About 30-60 minutes. | ||
#Obtain 2 '''control''' plates - these plates contain the same NGM lite medium described above and the bacterial strain on them are identical to your RNAi feeder strain, ''except'' that the pL4440 plasmid is only expressing RNA from the vector - it lacks DNA specific to any worm genes. | #Obtain 2 '''control''' plates - these plates contain the same NGM lite medium described above and the bacterial strain on them are identical to your RNAi feeder strain, ''except'' that the pL4440 plasmid is only expressing RNA from the vector - it lacks DNA specific to any worm genes. | ||
#Stack all 6 plates carefully (without tilting them) - put them in your lab section box | #Stack all 6 plates carefully (without tilting them) - put them in your lab section box with a labeled piece of your team color tape on top. | ||
# | #The plates will be stored at 4°C until you are ready to add worms. | ||
<br> | <br> | ||
'''4 days before next lab:''' | '''4 days before next lab:''' | ||
