HUM-MOLGEN archive: BIOT: May 1998

New BIOTs! The BIOT section is open for requests and offers of information regarding molecular biology and molecular genetics (protocols, techniques, products, collaboration, etc.). You can reach over 5016 of your colleagues, and on average you may expect up to twenty replies to a single message. This service is absolutely FREE of charge. Help yourself by helping your colleagues! - Please send high quality messages only, including full name, address and purpose. - Please use the appropriate TOPIC subject heading in your message. - Please state non-trivial questions only. - Please reply by private E-mail only, unless your request is of general interest to the entire HUM-MOLGEN community Messages may be refused without further notification. 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. Regards Dr. Raymond Clarke St. George Hospital r.clarke@unsw.edu.au ********************************************************************** 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 Sweden Tel: +46 (0)18 664266 (lab) / 664347 (writing-room) 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 help? 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 http://agriculture.tusk.edu/caens/genome/genome.html ********************************************************************** 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 skerfoot@acs.ucalgary.ca 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. Pathology St. Louis University Hospital 3635 Vista St. Louis MO 63110 USA phillinj@slu.edu 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 U.S.A. **********************************************************************