Hi, this is in respond to the question concerning tetrad analysis in pombe. Our lab does not have a sophisticated tetrad-pulling instrument. However, I am quite happy with the method that I've been using. The tools include: agar plates with considerable amount of moisture, a micromanipulator that can hold a solid glass needle of approximately 1-2 mm in diameter, glass needle of appropriate thickness at the tip (to be determined empirically). Usually I pour the plates on my bench and keep the lid closed to prevent overdrying. I use around 40ml of media for each standard petri dish therefore the thickness of the agar reaches around 1 cm. They are ready to use after 2-3 hours at room temperature. I then streak asci (sporulated diploid cells) across the plate in a straight line around 2 cm beneath the top of the plate. I then draw the tetrad array or matrix if you will underneath that straight line. A best illustration please see the handbook which I believe is titled "e!
xperiments in fission yeast" or something like that. The actual pulling of cells is hard for me to describe, short of physical demonstration. However, a few things to remember: 1) after you move the whole asci away from the line of cells, you have to give it some time for the cell walls to break down. Usually 1-3 hours at 36oC on YE plate is sufficient, however, I have also tried overnight on the bench (19oC) which also seems to work. Minimal media takes much longer. 2)rather than moving the cells by moving the needle, I find it easier to drag the cells across the plate by touch the cells slightly with the needle point and move the plate.
I hope these words will help. I might be of better assistance answering some more specific questions if they do come up. good luck!
Wenyi Feng
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