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To: HUM-MOLGEN@NIC.SURFNET.NL
Subject: BIOT:
From: Arthur Bergen <a.bergen@ioi.knaw.nl>
Date: Wed, 26 Aug 1998 12:05:07 MET
Organization: ioi.knaw.nl
Priority: normal

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
************************************************************************


   
 
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