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wiki:protocols:optogenetics

Protocol for optogenetics experiments

adapted from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6057767/, some of it directly! Do not use this text without modification outside of the lab.

Worm preparation for channelrhodopsin stimulation

Before the laboratory period, instructor prepares NGM plates with E. coli mixed with all-trans-retinal. All-trans-retinal (ATR) is a necessary cofactor for channelrhodopsin to function and is not naturally found in worms. To make E. coli with ATR, grow an overnight culture of 50 mL E. coli in LB. Centrifuge 50 mL culture at low speed, remove supernatant and resuspend cells in 10 mL M9 buffer (3.0 g KH2PO4, 6.0 g Na2HPO4, 5 g NaCl, 1 mL MgSO4 (1 M) per L H2O). This bacteria solution can be stored in the refrigerator for approximately one month. Immediately before making plates, add 1 μl 50 mM ATR (Sigma, all-trans-retinal, dissolved in ethanol) to 1 mL bacteria. Mix well by vortexing and pipet 50 μl per NGM plate. Allow to soak into plate (about 2 hours) in darkness because retinal is sensitive to light. For control ATR-minus plates, prepare the same way, except do not add retinal to the bacteria. Pick 20 to 40 L4 worms to each plate and incubate overnight (16–24 hours). Wrap plates in tinfoil or put in a dark box because both channelrhodopsin and retinal are sensitive to light.

In the laboratory period, students will measure the behavioral effect of light stimulation on each genotype of worms. First, students move worms from the ATR plates to NGM plates without food. There are two ways to do this. One, pick worms to an NGM plate without bacteria, then allow worms to crawl around plate some so that bacteria on the cuticle are rubbed off. Then use a pick without food to transfer worm to another NGM plate without food. This way there will be no bacteria on the final plate with worms. An alternative method is to pipet 300 μl of M9 buffer on to plate with worms, transfer with glass Pasteur pipet to another NGM plate, and blot excess liquid with a Kim-wipe. After transferring worms by either method, starve worms for 20–30 minutes. During this time, students can set up and learn how to use blue light stimulation.

Blue light stimulation

- check intensities from literature, but 1mW/mm2 should be a starting point.

wiki/protocols/optogenetics.txt · Last modified: 2021/02/18 05:48 by mscholz

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