Chemotaxis C.elegans are grow on NGM with OP50

Chemotaxis plates are
prepared with 10cm Petri dishes containing 10ml of assay agar (2% agar, 5mM KPO4
pH6, 1mM CaCl2, 1mM MgSO4). c.elegans have to be washed
twice in S-basal, once in distilled water and placed onto spot on the plate.
Mark two spots equidistant from center diameter on the bottom of petri dish,
mark them as A and B. Mark the spot X on the circumference of the petri dish
forming a triangle with A and B1µl of 1:100
benzaldehyde:EtOH is placed at test spot A and 1µl of ethanol was applied to
the other spot B. C.Elegans are added at spot X.For each spot, 1µl of 1 M NaN3
was applied to immobilize the worms when they reached the spot. After one hour of
chemotaxis, animals were counted, and Chemotaxis Index (CI) is calculated as
{number of animals at the test odorant A – number of animals at the solvent
B} / Total number of animals tested (A+B). Repeat the steps 2,3 with a
different concentration of 1µl of 1:1000 benzaldehyde:EtOHFor locomotor assay, take
C.elegans are grow on NGM with OP50 in nicotine-free plates. They are placed on
fresh plates 5-6 hours before the behavioral test. C.Elegans are then
transferred to plates with appropriate nicotine concentration. Observations are
recorded.First 10-12 minutes of the
test are discarded for results because the elegans have to get acclimated to
the new environment. The results give us insight of Acute Locomotor Assay.Elegans from step 3 are then
taken to measure the Locomotor Sensitization Assay. Transfer worms to a clean plate containing a lawn of OP50
and 100 ?M nicotine before transferring them to a second clean, nicotine-free
plate for an additional hour. Observe how the elegans
crawl on a plate covered in 40 ?L OP50 and a 10 ?M Nicotine concentration. For chemotaxis assays, we
used standard 10 cm Petri dishes containing 15 mL of an optimized nematode
chemotaxis medium 1.6% BBL agar; 1 mM MgSO4; 1 mM CaCl; 5 mM

KPO4. On test day, plates are allowed to warm on the bench to 20ºC, before
partitioning the outside of the dish into three sections – A,B and X for
attractant, control and middle section.

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Transfer about 50-150 L4
elegans to the chemotaxis plate by adding 10?L nicotine diluted in ethanol at A
and the control, ethanol diluent at B. We then placed the dishes
upside down and allowed to sit undisturbed for one hour.Observations are recorded at
the end of the hour. Chemotaxis index (CI) is calculated by counting the number
of elegans using the formula: (A-B)/(A+B+X) = CI.

 

 

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