DNA fibre FISH blues

Hans Dauwerse dauw at ruly46.medfac.leidenuniv.nl
Fri Jun 7 03:19:11 EST 1996


Hello Margaret
Lets share some experiences with Fiber FISH.
As you probably know here in Leiden the Halo technique was 
develloped. This is a good reproducible technique only it is 
very laborious. Therefore we tried the Dervish technique but 
this turned out to be very reproducible and one of the 
biggest disadvantages is that there are a lot of DNA breaks 
in the fibers giving rise to strange hyb results. DNA breaks 
are due to the force you put on the DNA after lysing the 
cells, for Dervish this is gravity, for the Fidlerova method 
this is the sliding of the coverslip.
We have develloped a new method which is reproducible but 
there is always some variation between the slides. You can 
first DAPI stain the slides and look at them defore using.
We use unfixed cells and a very gentle lysis method. We 
found out that coated slides are essential, APES gives the 
best results, else you lose to much DNA during the 
hybridization procedures. Denature the slides only 1-2 min..
Another striking observation is that certain areas on the 
slides give the nicest fibers in most cases at the edges, so 
try to probe the entire slide.
For your point 2), this is difficult to answer. As we have 
quite a lot of experience with FISH it did not really take 
long to get things working. Adjusting protocols to our needs 
took us a couple of month. 
For point 3). Thats were you use it for, to find out new 
things.
I allready answered point 4.
If you are interested in the method you can email me.

Hans



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