probe size in FISH on metaphase chromosome

[Zhenghui Liu]

I currently want to use the method of fluorescence in situ hybridization to
locate which allele of the pair of chromosomes of ES cell was targeted by my
targeting vector because I had a heterozygote of ES cell, in which one
allele of
another
gene had been targeted, and I have to know if my new targeting event
occurred in the same chromosome or not.  In my pre-experiment, I tried two
probes on metaphase chromosome spreads.  Both should be positive because
they have the same origin with chromosome.  I tried once, but failed without
signals except high background.  The probe size I used is that one is about 3kb
and the other is about 0.6kb.  I cloned both of them in cloning vectors and
labeled them using the whole plasmid (insert + Vector).  Before I try again, my
questions are below.
1.  How long is the minimum size of a probe applied in FISH on metaphase
chromosome?  Does 0.6kb work well?
2.  How can I reduce the background?  Don't forget I use mouse chromosome.
3.  Is there a good book for all aspects of FISH?
My e-mail is liu at omrf.ouhsc.edu.
Thanks a lot.



Zhenghui Liu


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