test and reply to tetrad analysis in pombe
Carl at SINCO.demon.co.uk
Fri Mar 31 11:42:24 EST 2000
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!
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.
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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