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Subject: BIOT: May 1998
From: Edward Wilcox <edwilcox@pop.nidcd.nih.gov>
Date: Wed, 3 Jun 1998 13:44:58 -0500

New BIOTs!

The BIOT section is open for requests and offers of information regarding
molecular biology and molecular genetics (protocols, techniques, products,
collaboration, etc.).

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Edward Wilcox
Trevor M. D'Souza
(BIOT editors)

This BIOT message contains:

1)  How do I enhance chromosomal FISH analyses?
2)  How can I improve the reproducibility of the non-radioactive detection?
3)  Looking for a protocol to isolate genomic DNA from blood clots.
4)  Need information on efficiently sequencing plasmid-based templates.
5)  Does anyone know of a mammalian expression vector containing SEAP?
6)  Does anyone know about expression of IRBP-promoter driven transgenes?
7)  A question on the usefulness of commercially available cDNA arrays.


We would appreciate an improved method for using total YAC/yeast DNA in
chromosomal FISH analyses. To date we do not get an unambiguous signal using
total DNA. Any tricks to improving this signal to a satisfactory level before
Alu PCR or YAC isolation would be very much appreciated.


Dr. Raymond Clarke
St. George Hospital


Dear colleagues:

I am using Boeringer Mannheim's non-radioactive digoxigenin based system for
northern blots. It works quite efficiently, but from time to time I get high
background or low signal. I would be happy to get in contact with people also
working with the system to share ideas of how to improve the reproducibility of
the detection.

Jonas Byström
Department of Medical Science
Clinical Chemistry
Entr. 60
University Hospital
S-751 85 Uppsala

Tel: +46 (0)18 664266 (lab) / 664347
Fax: +46 (0)18 663703
E-mail: jonas.bystrom@klinkem.uu.se


I am looking for a protocol to isolate genomic DNA from blood clots.  Can anyone

Partha P. Majumder
Anthropology and Human Genetics Unit
Indian Statistical Institute
203  B.T. Road
Calcutta  700 035

E-mail: ppm@isical.ac.in


We are sequencing plasmid-based templates using  dRhodamine dye-terminator
chemistry but are interested in using the Big-dye terminators. Can somebody
please give us some information on dilutions (and protocol) used to efficiently
sequence using a fraction of the ABI recommended reagent volume.

Please reply to edsmith@acd.tusk.edu

Thank you.

Ed Smith

Assoc. Professor
Animal Genetics


We are trying to build a protein fused to secreted alkaline phosphatase.  Does
anyone know of a mammalian expression vector containing SEAP and where it might
be obtained?  There is one vector that would be appropriate, called "APtag-1".
Has anyone heard of it?

Thanks very much,

Steve Kerfoot
Department of Clinical Neurosciences
University of Calgary, Faculty of Medicine
3330 Hospital Drive N.W.
Calgary, Alberta, Canada T2N 4N1


Using the IRBP-promoter (interphotoreceptor retinol binding protein) I have
generated 3 independent lines of transgenic mice that express in ONL
photoreceptor cells and the pineal gland. Both lines also show evidence for
transgene activity in the anterior lobe of the pituitary gland, where it is not
known to be expressed as far as I am aware.

1) Does anyone know whether there is any ontological relationship between
photoreceptor cells in the retina and the pituitary gland in mice (e.g. whether
these cell types share a common precursor) that may explain this finding?

2) Does anyone know of other examples where IRBP-promoter driven transgenes were
also expressed in the anterior pituitary?

Marc Vooys

The Netherlands Cancer Institute Div. of Molecular Genetics, Plesmanlaan 121
Tel  : +31 20 5121994 1066 cx Amsterdam
fax  : +31 20 5122011 The Netherlands
email: mvooys@nki.nl

Has anyone used one of the commercially available cDNA arrays from
Clontech, Research Genetics, or Genome Systems? How about their
accompanying computer programs for analysis of phosphorimager files? Is
there any definite advantage to either lysed colonies or PCRed DNA (ests)
in terms of quality of hybridization, amount of background, etc? My
proposed uses would be 1. Analysis of primary tumors 2. Analysis of paired
cell lines (plus or minus drug).

Thanks for any information.
Nancy Phillips, M.D.
St. Louis University Hospital
3635 Vista
St. Louis MO 63110 USA
314-577-8782, fax 314-268-5120

Trevor M. D'Souza, Ph.D.
Research Associate
Department of Microbiology
Michigan State University
East Lansing, MI 48824-1101

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