User:Nadiezda Fernandez-Oropeza/Notebook/Notebook/2011/05/27

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More corrections

  • Andy’s last day in the lab

As it was his last day in the lab, he looked over my shoulder as I completed most of the steps in the motility assay. I am really glad he did because he found “little” mistakes that at the end have a great impact on the results. Actually, one of them, or the combination of all of them might have been causing the clusters, or the extreme brightness in the microtubules.

  • The general rules are:
    1. Keep everything cold at all times. Reduce to the minimum possible the amount of time that you expose the chemicals to room temperature.
    2. Be extremely careful when handling not only the chemicals but also the tubes, pipettes, tips, etc. This implies not leaving the tubes opened on the racks, attaching firmly the tips to the pipettes, etc.
    3. Avoid bubbles when pipetting. A good way of doing this is to pull the tip out of the liquid surface as the last drop is being poured. Also, it is important not to blow the volume completely out of the tip when mixing chemicals.
  • As far as the motility assay process, some things to be corrected are:
    1. The Taxol in DMSO solution has to be vortex and centrifuged every time before using it.
    2. Most importantly, when preparing the 29% Rhodamine-labeled I should use the medium sized tubes and not the smallest ones because the medium ones fit correctly in the thermal cycler, allowing the contact between the lid of the thermal cycler and the lid of the tube.
    3. Something to consider too is that I prepared new TSB. Just as a reminder, the GTP comes in a jar. Usually the chemical is all scattered on the inner surface of the jar. Therefore, one has to handle it really carefully. When adding the 1.06x PEM make sure that the tip doesn’t touch the walls of the jar, and deposit the liquid directly in the bottom. Once this is done, vortex it really well and transfer it to a vial. Glycerol can give a lot of trouble; the best way to pipette is to use the 1000 µL pipettor, despite the fact that we will be collecting 120 µL of glycerol.
    4. For the preparation of the tubulin suspensions, doing things as fast and carefully as possible is critical. Ideally, the solution should be placed at the bottom of the tubes and splashes to the walls should be avoided.
  • For the microscope:
    1. Have the mercury lamp turned on at least half an hour before collecting data and make sure that everything is placed correctly on the microscope. After finishing, make sure to clean the oil from the objective.



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