advice on digoxigenin in situ's

TurnerG TurnerG at STARBASE1.CALTECH.EDU
Wed Jun 9 03:59:59 EST 1993


Fellow arabidopsis workers-
   We would really appreciate some advice on nonradioactive in situ's.
We are trying to get digoxigenin labelled (boehringer genius kit) in situ
hybridizations to work.  We are currently using a protocol from Vivian Irish,
with an ap3 probe (relatively high abundance message) on landsberg erecta 
flowers, but we have run into a couple problems:

	1-  high background staining-  the staining lacks specificity, and is rather
dark all over.
	2-  low background, but signal extremely weak
	3-  there is a great deal of variability in the staining which is observed.
ie. we sometimes see both problems 1 and 2 on different slides which underwent
the procedure together.  Occasionally we also see variability on the same
slide, ie. there may be high signal from the stamens in one slice, but not in
the adjascent section.  

Basically, we need to know what variables decrease background, and which
parameters might be responsible for low levels of signal so we can tinker with
them.  I would also like to know what is responsible for the variability we
observe, if anyone knows.  Any suggestions you have would be much appreciated,
we'll post them on the network when we get them.  Also, if you have a protocol
which works well, you could fax it to us at (818) 449-0756. 

Thanks A lot
Glenn Turner & Zhongchi Liu

turnerg.ccomail at starbase1.caltech.edu



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