HUM-MOLGEN archive: BIOT:

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) From: Edward Wilcox <edwilcox@pop.nidcd.nih.gov> ------------------------------------------------------------------- This BIOT message contains: 1) A need for help problem-solving using the ABI 377 sequencer. 2) How does one sample drinking water using male-specific bacteriophage as an indicator? 3) A collaborative request to use green fluorescent protein (GFP) as a reporter gene in cell lines. 4) What is the best way to obtain a complete sub-clone library from a YAC? 5) High school students in the UK would like to conduct DNA amplification experiments. 6) What are convenient sources of healthy B and T cell lymphoma lines? 7) A Ph.D. student is requesting help with prenatal diagnosis for chromosomal abnormalities. ******************************************************** Dear Colleagues, I am writing to see whether any of you have ever used a protocol for genomic DNA amplification. A colleague passed a protocol to us. This is a PCR-based protocol using a random 15-mer primer to produce large quantities of DNA from a small amount of genomic DNA (Original reference: Zhang et al. 1992. PNAS, 89:5847-5851). We could not see the amplified higher molecular weight DNA on the agarose gel after the reaction. Nor could we see any improvement in PCR using the amplified genomic DNA compared with the non-amplified DNA. We tried several times with 15-mers ordered at different times. The second question is: Is anyone doing automated genotyping using an ABI 377 sequencer? We are having problems with green markers. When we started genotyping, the results were excellent. But now blue color is leaking into green. In other words, the blue peaks are also present as green peaks. This has resulted in heavy background in green markers, making the Genotyper analysis more difficult. ABI suggested making a new matrix standard. We did but after a few gels, we have to do it again. It is time consuming. Does anyone have the same experience and/or any better ideas? Hongrun Yu Dept. of Biochemistry Wake Forest Univ. School of Med. Winston-Salem NC 27157 (336) 716-3176 E-mail: hyu@BGSM.EDU *************************************************** I need to test some drinking water samples for contamination using the presence of male-specific bacteriophage as an indicator. Does anyone have experience with this type of analysis and/or know of a source for male-specific phage antibodies? Thank you in advance. Susan Jenkins, Ph.D. Plant and Microbial Biology Department 211 Koshland Hall University of California Berkeley, CA 94720-3102 Phone (510) 643-1968 Fax (510) 642-4995 E-mail: sjenkins@nature.berkeley.edu ************************************************* I am writing to the list in the hope that someone may be able to offer me some advice regarding the use of Green fluorescent protein (GFP) as a reporter gene in human (& other mammalian) cell lines such as HepG2 cells. I am hoping to use GFP, together with a GFP variant (blue or yellow), to normalize for transfection efficiency in transient transfections in 96 well culture plates, as a reporter of gene transcription from promoter regions I wish to study. If anyone has any experience of this, or problems associated with this approach I would be very interested. Yours sincerely Malcolm S. Ogg Dr. Malcolm S. Ogg Centre for Cardiovascular Genetics Department of Medicine UCLMS The Rayne Institute 5 University Street London WC1E 6JJ Tel: 0171 209 6977 Fax: 0171 209 6212 Email: rmhamso@ucl.ac.uk *************************************************** Dear HUM MOL-GEN subscribers: I am presently subcloning from YACs. I would like advice on the best way to obtain a complete subclone library from a YAC. Do I need to isolate the YAC by PFGE and if so, could this be complicated by the size of the YAC in relation to full length Yeast chromosomes or complicated by low YAC copy number? I would also like information on how to subclone just the ends of the YAC. I would like to know how to obtain the vector 'pYAC2PAC-1' that has been described for subcloning the ends of YACs via recombination from spheroblasts. Regards Dr. Raymond Clarke St. George Hospital E-mail: r.clarke@unsw.edu.au ************************************************ Can the human genetics research community out there help UK high school students conducting DNA amplification experiments? Relatively large amounts of a suitable DNA size marker are required at low cost. The amplified DNA product is about 500 base pairs long which students run out on a small 2% agarose gel. Safety concerns effectively rule out students staining gels with Ethidium Bromide and less sensitive stains require at least 2ug per lane of a marker such as Phi X174 HaeIII to be clearly visualized. Does anyone have a simple protocol for prepping PhiX174, since commercially it is far too expensive, or even some better ideas for DNA size markers? Ian Muchamore Project Manager, Medicine in Society Programme The Wellcome Trust 210 Euston Road, London NW1 2BE Tel 0171 611 8636 Fax 0171 611 8269 E-mail: i.muchamore@wellcome.ac.uk Web: http://www.wellcome.ac.uk ********************************************************* I am looking for a convenient source of healthy B and T cell lymphoma lines such as MMOLT or Jurkat. Jay Stoerker Genzyme Genetics Framingham MA 01701 Phone: 508-271-2634 E-mail: Jstoerker@Genzym.com ************************************************ Dear Colleagues, I am a Ph.D. student working on prenatal diagnosis for chromosomal abnormalities. Advice/Suggestions on the following will be highly appreciated. 1. Chorionic Villus: To improve index and quality of metaphases suitable for banding. 2. Cord Blood: Sensitive methods to check the presence of maternal cells contamination. 3. Amniotic fluid: To improve growth of amniotic fluid cell cultures. DR. VAIDEHI JABANPUTRA Kindly reply at the following email address: vjobanputra@hotmail.com ******************************************************* ************************************************************************ Dr. Arthur A.B. Bergen Department of Ophthalmogenetics The Netherlands Ophthalmic Research Institute (IOI) Royal Academy of Sciences of the Netherlands (KNAW) ** Snail-mail: ** ** FAX: ** ** E-mail: ** P.O.Box 12141 (+31)206916521 A.Bergen@IOI.KNAW.NL 1100 AC Amsterdam The Netherlands ************************************************************************ ************************************************************************ Dr. Arthur A.B. Bergen Department of Ophthalmogenetics The Netherlands Ophthalmic Research Institute (IOI) Royal Academy of Sciences of the Netherlands (KNAW) ** Snail-mail: ** ** FAX: ** ** E-mail: ** P.O.Box 12141 (+31)206916521 A.Bergen@IOI.KNAW.NL 1100 AC Amsterdam The Netherlands ************************************************************************