In article <8c1n7s$569$1 at mercury.hgmp.mrc.ac.uk>, Wenyi Feng
<wfeng at newssun.med.miami.edu> writes
>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
Right. Specific question:
It is not the simple dissection of fission yeast tetrads that I am
looking for advice on, but the ORDERED dissection of the ascospores. As
the spores lie in the ascus they are in a certain order. As the ascus
degenerates the spores tend to pull together by surface tension and the
order is lost. I want to place spores on a grid in the order in which
they lie in the tetrad. It may not be possible.
Carl Singer
--
Carl Singer
Singer Instruments
Nothing is impossible..as long as you get someone else to do it.
Please do not email me at this address, use yeast at singerinst.co.uk.
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