test and reply to tetrad analysis in pombe

Carl Singer 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!
>
>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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