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    Worm Bleaching (Synchronized Population Starting With Eggs)

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    agbag12
    The document provides instructions for propagating C. elegans worms from eggs through bleaching and washing steps. The worms are first harvested from plates using water and centrifuged. NaOH and bleach are added to the tube to bleach the worms, releasing eggs. The tube is centrifuged and washed multiple times with M9 media to remove the bleach. The number of eggs is then counted under a microscope. Finally, enough eggs are pipetted onto plates with bacteria food to achieve the target number of eggs.

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    Worm Bleaching (Synchronized Population Starting With Eggs)

    Uploaded by

    agbag12
    22 views2 pages
    The document provides instructions for propagating C. elegans worms from eggs through bleaching and washing steps. The worms are first harvested from plates using water and centrifuged. NaOH and bleach are added to the tube to bleach the worms, releasing eggs. The tube is centrifuged and washed multiple times with M9 media to remove the bleach. The number of eggs is then counted under a microscope. Finally, enough eggs are pipetted onto plates with bacteria food to achieve the target number of eggs.

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    Procedure on how to bleach C. elegans worms

    Original Title

    Worm Bleaching

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    The document provides instructions for propagating C. elegans worms from eggs through bleaching and washing steps. The worms are first harvested from plates using water and centrifuged. NaOH and bleach are added to the tube to bleach the worms, releasing eggs. The tube is centrifuged and washed multiple times with M9 media to remove the bleach. The number of eggs is then counted under a microscope. Finally, enough eggs are pipetted onto plates with bacteria food to achieve the target number of eggs.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as DOCX, PDF, TXT or read online from Scribd
    Flag for inappropriate content
    Download as docx, pdf, or txt
    22 views2 pages

    Worm Bleaching (Synchronized Population Starting With Eggs)

    Uploaded by

    agbag12
    The document provides instructions for propagating C. elegans worms from eggs through bleaching and washing steps. The worms are first harvested from plates using water and centrifuged. NaOH and bleach are added to the tube to bleach the worms, releasing eggs. The tube is centrifuged and washed multiple times with M9 media to remove the bleach. The number of eggs is then counted under a microscope. Finally, enough eggs are pipetted onto plates with bacteria food to achieve the target number of eggs.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as DOCX, PDF, TXT or read online from Scribd
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    Worm Bleaching (Synchronized population starting with eggs) Worms will be propagated using OP-50 prior to lab.

    The prep staff will harvest the worms by washing the surface of the plate with 3 ml of deionized water. 1. 2. At the TA bench you should find a microcentrifuge tube with a 775 l aliquot of worms. Put on a lab coat, goggles, and gloves. Add 25 l of 10M NaOH and 200 l of bleach to the tube. Next, cap the tube and mix by inverting constantly for 4 minutes. It is crucial that this step is not done for more than 4 minutes as the NaOH/bleach mixture will kill the eggs. Quickly spin the tube in a microcentrifuge at 3,500 RPM for 1 minute. Quickly remove 850 l of the supernatant, being careful not to get too close to the bottom of the tube where the microscopic translucent eggs reside. Add 850 l of M9 media and mix by inverting for 5 seconds, followed by centrifugation at 3,500 RPM for 1 minute. Quickly remove 850 l of the supernatant, being careful not to get too close to the bottom of the tube where the microscopic translucent eggs reside. Add 850 l of M9 media and mix by inverting for 5 seconds, followed by centrifugation at 3,500 RPM for 1 minute. Remove 850 l of the supernatant, being careful not too get to close to the bottom of the tube where the microscopic translucent eggs reside. You will need to check for the quantity of eggs remaining in the tube using a microscope. Flick the tube gently with your finger, pipet up and down several times with a P200 pipetman set to 100 l and transfer 10 l (with a P20 pipetman) to a microscope slide (within the divot/indentation of the slide). Retrieve a compound microscope from under the bench in front of you. Check the eggs, first under 4X and then under the 10X objective. Determine approximately how many eggs you have in 10 l.

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    You want approximately 20 eggs on a 100mm petri dish containing the appropriate bacteria. You may need to adjust the volume of M9 media in order to conveniently pipet the proper amount to achieve the 25 egg target. Apply the drop of eggs to an area outside of the area containing the bacteria (see below).

    12. Place the plate on a tray on the TA bench for your section. The plates will be kept in an 15C incubator.

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