HDTenEightyP/NetNews huggingface dataset:
From: kristoff@NET.BIO.NET (Dave Kristofferson)
Newsgroups: bionet.molbio.bio-matrix
Subject: Re: Bio Matrix conference.
Date: 16 Dec 92 21:27:48 GMT
Distribution: bionet


> I continue to develop my oncogene database, and I am very interested
> in these issues.  I would very much like to do my work within the
> context of standards.  In fact, one of the more frustrating problems
> with my database has been the struggle to *FIND* standards; e.g. for
> gene names.  (I have good reason to believe that this is not trivial.)
> I would like to take this opportunity to initiate a discussion on the
> standards available for storing biological data (e.g. the kind of
> stuff that is in the FEATURES part of Genbank).
> 
> If there is any interest, I would be delighted to post a description
> of my database for public examination.
> 
> -- 
> David Steffen

David,

	Part of NCBI's charter, according to my possibly incorrect
understanding, is to guide these efforts.  It would seem to be a good
idea if people who are going to establish new databases (and continue
to maintain older ones), took the time to meet with them and hash
these things out.  I am not sure what their formal policy is on this
and whether or not there is a specific contact person for these kind
of issues, but hopefully someone from NCBI can comment on this in this
forum.

Dave Kristofferson
From: PARSONS_A@snd01.pcr.co.uk
Newsgroups: bionet.molbio.bio-matrix
Subject: Use of Internet by Industrialists
Date: 17 Dec 92 10:12:53 GMT
Distribution: bionet
Original-To: BIOMATRX <BIOMATRX@UK.AC.DARESBURY>

Bioneteers,

I am forwarding this on behalf of another board.

Tony Parsons - Pfizer Central Research (UK) parsons_a@snd01.pcr.co.uk

==============================================================================
From: Thomas J Cozzolino <RBNTJC@COM.ROHMHAAS.ROHVM1>
Subject: Industry Use of Internet?
To: Multiple recipients of list CHMINF-L <CHMINF-L@EDU.INDIANA.UCS.IUBVM>

Folks,

Our company is in the process of gathering information on Internet
usage by other companies, especially other chemical companies.

o  Is anyone willing to share their experiences, successes, and
   usage patterns regarding the Internet?

o  What are the value-added benefits of using the Internet for
   your Research and Business?

o  What are the biggest challenges facing you, the providers of
   Internet services?

o  How difficult has it been to change your particular corporate or
   Information Technology / Information Services cultures to embrace
   this "brave new world" view?

Any and all help is appreciated.  I will post a summary if there is
interest.  Also, if I should post this to other newsgroups, that
information is also much appreciated.

Thanks for reading.  Please reply directly if possible, or to this
newsgroup of you prefer.
+============================================+
|   Thomas J. Cozzolino - Rohm and Haas Co.   =
|   InterNet:    tcozz@rohmhaas.com            =
|                                               =
|     Internet Access for Everyone..             =
|         Isn't it time?                          =
|                                                  =
|   - Opinions expressed are my own, not            =
|     necessarily those of Rohm and Haas Company     =
+=====================================================+
From: shibumi@NET.BIO.NET ("Kenton A. Hoover")
Newsgroups: bionet.molbio.bio-matrix
Subject: Test message
Date: 17 Dec 92 19:04:51 GMT
Reply-To: shibumi@net.bio.net
Distribution: bionet

A test message

| Kenton A. Hoover                                 |      shibumi@net.bio.net |
| BIOSCI Network Administrator                     |                          |
| BIOSCI/IntelliGenetics, Inc.                     |          +1 415 962 7300 |
|=========== net.bio.net -- The New Home of the bionet Newsgroups ============|
|         The only thing necessary for the triumph of evil is for good        |
|                men to do nothing.  -- Edmund Burke (1729-1797)              |
Newsgroups: bionet.molbio.bio-matrix
From: gilbertd@sunflower.bio.indiana.edu (Don Gilbert)
Subject: Re: Beyond GenBank.  
Organization: Biology, Indiana University - Bloomington
Distribution: bionet.molbio.bio-matrix
Date: Tue, 15 Dec 1992 02:46:27 GMT

What you suggest, about cross-referencing studies on the function
of a sequence to the genbank entry, is at least partly addressed
in NCBI's Entrez CD-Rom, which includes not only Genbank but the
related Medline article abstracts.  You should be able to, given
a genbank entry, find many of the journal articles which discuss
this sequence, by using Entrez' software for searching out neighboring
articles and entries.   You can find more info on Entrez thru ftp
to ncbi.nlm.nih.gov, including order forms.        

-- Don

-- 
Don Gilbert                                     gilbert@bio.indiana.edu
biocomputing office, biology dept., indiana univ., bloomington, in 47405
Newsgroups: bionet.molbio.bio-matrix
From: brianf@dna.uvm.edu (Brain Foley)
Subject: Re: Beyond GenBank.  
Organization: Division of EMBA, University of Vermont
Distribution: bionet.molbio.bio-matrix
Date: Mon, 14 Dec 1992 22:51:30 GMT

	While GenBank does a fine job of storing the sequences of DNA
and RNA that have been experimentally determined, it does not do so
well at storing information that has been determined about HOW these
sequences function in cells.  For instance, there might be one
publication that describes the sequence of a new bacterial promoter.
GenBank will enter the sequence into the database, along with
information about the seuence from that publication.  Howerver, if six
more publications follow this one, providing more information abut
what proteins bind to different regions of the promoter, it is not
GenBank's responsibility to enter that data.  Likewise GenBank has the
human ras oncogene in it, but does not have information about how many
human tumors have been found to have mutations in the ras oncogene.
The papers describing mutations in the ras oncogene generally only
report the mutation and perhaps show a few base pairs near the
mutation, rather than the sequence of the whole gene.

	Thus there is a wealth of information about DNA sequences that
is not getting into GenBank.  Most of this information does not even
truly belong in GenBank, but rather in a secondary database devoted to
the topic at hand.  Should we have a database of human mutations,
with information about whether they are germ-line mutations or
mutations that developed within somatic cells of an individual,
information about the phenotype of the mutated cells/individuals, and
so on?

	Such new databases should at least be cross-indexed to GenBank
so that we can call up the GenBank entry for the gene.  Conversely if
we are looking at the GenBank entry for the ras oncogene, it would be
nice to see that there is a separate database devoted to mutations
found in this gene.

	What is the current state of the art in creating new databses
that enhance GenBank?
--
********************************************************************
*  Brian Foley               *     If we knew what we were doing   *
*  Molecular Genetics Dept.  *     it wouldn't be called research  *
*  University of Vermont     *                                     *
********************************************************************
From: shibumi@NET.BIO.NET ("Kenton A. Hoover")
Newsgroups: bionet.molbio.bio-matrix
Subject: Test message
Date: 17 Dec 92 19:11:22 GMT
Reply-To: shibumi@net.bio.net
Distribution: bionet

Test message #2 is created.

| Kenton A. Hoover                                 |      shibumi@net.bio.net |
| BIOSCI Network Administrator                     |                          |
| BIOSCI/IntelliGenetics, Inc.                     |          +1 415 962 7300 |
|=========== net.bio.net -- The New Home of the bionet Newsgroups ============|
|   All Governments, including the worst on earth and the most tyrannical on  |
|   earth, are free Governments to that portion of the people who voluntarily |
|   support them.                                                             |
|       -- Lysander Spooner                                                   |
From: steffen@mbcr.bcm.tmc.edu (David Steffen)
Newsgroups: bionet.molbio.bio-matrix
Subject: Re: Bio Matrix conference.
Date: 16 Dec 92 21:19:12 GMT
Distribution: bionet
Organization: Baylor College of Medicine, Houston, Tx
NNTP-Posting-Host: mbcr.bcm.tmc.edu


brianf@dna.uvm.edu (Brain Foley) writes:
>At the Biomatrix conference in Waterville Valley New Hampshire a
>couple years ago, GenBank was hardly mentioned.  All sorts of other
>biological databases were talked about.  One person had a database of
>all the legumes in the world (where they grow, what they look like,
>what soil type they live in...) another person had a database of video
>images of sea life, the types of databases were incredibly diverse.

kristoff@net.bio.net (David Kristofferson) writes:
>Part of the problem is also that many people want to go off and
>reinvent the wheel by setting up their own database instead of working
>with existing organizations.  After all, it is so much easier and less
>painful to invent one's own nomenclature, etc., than to have to fly to
>an international standards committee meeting and sit (half-asleep due
>to jet lag) around a table and argue about the meaning of words.
>However, barring such efforts, one is left with the kind of problems
>that Tom always rages at.

I continue to develop my oncogene database, and I am very interested
in these issues.  I would very much like to do my work within the
context of standards.  In fact, one of the more frustrating problems
with my database has been the struggle to *FIND* standards; e.g. for
gene names.  (I have good reason to believe that this is not trivial.)
I would like to take this opportunity to initiate a discussion on the
standards available for storing biological data (e.g. the kind of
stuff that is in the FEATURES part of Genbank).

If there is any interest, I would be delighted to post a description
of my database for public examination.

-- 
David Steffen
Department of Cell Biology, Baylor College of Medicine, Houston TX 77030
Telephone = (713) 798-6655, FAX = (713) 790-0545
Internet = steffen@bcm.tmc.edu
Newsgroups: bionet.molbio.bio-matrix
From: toms@fcs260c2.ncifcrf.gov (Tom Schneider)
Subject: Re: Re: Beyond GenBank.
Organization: Frederick Cancer Research and Development Center
Distribution: bionet.molbio.bio-matrix
Date: Thu, 17 Dec 1992 02:07:37 GMT

>*  Brian Foley               *     If we knew what we were doing   *
In article <BRIANF.92Dec16105446@dna.uvm.edu> brianf@dna.uvm.edu (Brain Foley) writes:

(Hmm.  Sorry, but your news reader is calling you "Brain"...)

Brian pointed out a number of the difficulties of duplications in the database.

Here's a way around them.  Think years into the future.  We will then have a
complete human sequence.  So in that set of chromosomal sequences there WILL be
many partially duplicated things (like ALU sequences), but they are all part of
the normal set of sequences.  So I suggest that we attempt to stick to that
natural model.  When another person is sequenced, that is recorded either as
changes from the first (ie a form of compression) or as a separate example
sequence.  With the appropriate software, extraction would give either
sequence as if it were whole.

Now, the person who wants all the cytochrome C sequences can write a program
which specifies which small fragments to extract from the large Library of
sequences.  This allows the data to be stored in a nice continuous natural
form, but also to be manipulated for whatever purposes we like.  I developed a
language to do this:

  DEoxyribonucleic
    LIbrary
      LAnguage

  DELILA

(Unfortunately I didn't think about RNA genomes, but the name NULILA is
not as nice!!  Also, Delila is somewhat out of date, but the general
idea is still valid.)

Folks interested can refer to:

@article{Schneider1982,
author = "T. D. Schneider
 and G. D. Stormo
 and J. S. Haemer
 and L. Gold",
title = "A design for computer nucleic-acid sequence storage, retrieval and
manipulation",
journal = "Nucl. Acids Res.",
volume = "10",
pages = "3013-3024",
year = "1982"}

@article{Schneider1984,
author = "T. D. Schneider
 and G. D. Stormo
 and M. A. Yarus
 and L. Gold",
title = "Delila system tools",
journal = "Nucl. Acids Res.",
volume = "12",
pages = "129-140",
year = "1984"}

and can find the programs in the archive at ncifcrf.gov/pub/delila.

Also, a lot of these issues can be focused by having good documentation.  I
have a draft of a paper on such a Philosophy for GenBank in the same archive:
            philgen.ascii.Z philgen.ps.Z
I just made changes today.  Please pick up a copy and feel free to comment.
Good comments will be incorporated, with your permission.

  Tom Schneider
  National Cancer Institute
  Laboratory of Mathematical Biology
  Frederick, Maryland  21702-1201
  toms@ncifcrf.gov
Newsgroups: bionet.molbio.bio-matrix
From: toms@fcs260c2.ncifcrf.gov (Tom Schneider)
Subject: Re: Beyond GenBank.  
Organization: Frederick Cancer Research and Development Center
Distribution: bionet.molbio.bio-matrix
Date: Wed, 16 Dec 1992 02:32:06 GMT

In article <BzA51G.4vD@usenet.ucs.indiana.edu>
gilbertd@sunflower.bio.indiana.edu (Don Gilbert) writes:

>What you suggest, about cross-referencing studies on the function
>of a sequence to the genbank entry, is at least partly addressed
>in NCBI's Entrez CD-Rom, which includes not only Genbank but the
>related Medline article abstracts.  You should be able to, given
>a genbank entry, find many of the journal articles which discuss
>this sequence, by using Entrez' software for searching out neighboring
>articles and entries.   You can find more info on Entrez thru ftp
>to ncbi.nlm.nih.gov, including order forms.        

If you really believe this, then either you've never done a serious
project - in which you spent months at this kind of work - or you
will be willing to set up a collaboration with me....

You would get completely bogged down for days with just CRP.

  Tom Schneider
  National Cancer Institute
  Laboratory of Mathematical Biology
  Frederick, Maryland  21702-1201
  toms@ncifcrf.gov
From: GIETZ@bldghsc.lan1.umanitoba.ca
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: site directed mutagenesis problems
Date: 14 Dec 92 23:38:00 GMT
Distribution: bionet

I realize this may be a bit late but our mail port was not working 
these messages were returned : D.G.

---------------------fm writes----------------------------------------
I recently posted the problems I had and appreciate your
responses.  However, I am still not getting the mutants using
the Bio-rad kit.  I am getting transformants with the negative
control DNA - where I make the second strand in the absense of
any mutagenic primer.

Just recently, I found that my SS DNA was able to transform XL1
blue competents.  Apparently the SS DNA  prep was contaminated
with DS DNA.  Has anyone had experienced this problem?  I am
making SS DNA from the Bluescript plasmid in CJ236 cells.  I am
thinking of gel purifying the SS DNA.  Is this a logical or a
stupid idea?  I appreciate any comments.

--------------------------------------------------------------------------
I have had similar problems with another strain and got rid of contaminating
DS DNA and RNA by treatment of supernatant with DNase I 10U/ml
and RNase A 10ug/mL for 15 min at 37 degrees C.  Do this before PEG
pptting your phage and your ssDNA prep will be very clean!

Dan Gietz
University of Manitoba
GIETZ@BLDGHSC.LAN1.UMANITOBA.CA

Newsgroups: bionet.molbio.methds-reagnts
From: tronni@nphi.fi
Newsgroups: bionet.molbio.methds-reagnts
Subject: Thanks for random priming advice
Date: 16 Dec 92 23:00:08 GMT
Organization: National Public Health Institute, Finland

Thanks to all friendly fellow-netters who gave me random priming
advice. I'll cut DNA amount to 25 ng and incubate overnight at RT
from now on (with a new batch of Klenow!). 

Cheers,

Tapani Ronni
National Public Health Institute
Mannerheimintie 166
00300 Helsinki
FINLAND
From: djt2@po.CWRU.Edu (Dennis J. Templeton)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: electroporation (to make cDNA libraries)
Date: 14 Dec 1992 22:01:04 GMT
Organization: Case Western Reserve University, Cleveland, OH (USA)
Reply-To: djt2@po.CWRU.Edu (Dennis J. Templeton)
NNTP-Posting-Host: slc12.ins.cwru.edu


In a previous article, pakapke@IASTATE.EDU () says:

>
>     We would like to get away from in vitro packaging our phage DNA and
>simply electroporate it into the cell directly.  Anyone in net-land had
>experience doing this?  Are there any references published using this
>technique to produce a phage library?
>

Hmm.  I don't have a direct answer for you, but I would be cautious about
trying to electroporate DNA as large as the concatemers of lambda you get
after ligation.

On the other hand, we spent months making a good cDNA library out of
LambdaZAP, THEN got experienced in electroporation of coli.  Given the high
electrocompetency rate (10e10 is not unrealistic) it seems we might have
been just as successful at making our cDNA library in pBluescript.

Here are some numbers:

5 ug A+ RNA, makes about 5 ug DS cDNA
Ligating 1 ug of this into 0.5 ug of *prepared* (see below) pBluescript
	should result in about 0.1 ug of circularized vector (probably
	more)
Electroporation of this (in several batches) has the potential to generate
10e8 to 10e9 clones.  

From a practical standpoint, the high electroporation efficiencies are at
very low levels of supercoiled plasmid.  We have never gotten more than
10e6 clones from a single pulse, but 10 pulses of a ligation are practical,
and 10e6 is plenty of cDNA's anyway.

Input from any who has made libraries this way would be appreciated, and
those contemplating it should look at the article in this month's
BioTechniques (13, Nr6. 862-864, 1992) wherein the authors describe using
pBluescript to make T-tailed vector to clone cDNA's a al Okayama and Berg.

good luck,

dennis
From: GIETZ@bldghsc.lan1.umanitoba.ca
Newsgroups: bionet.molbio.methds-reagnts
Subject: RE: MC1061/P3 minipreps
Date: 14 Dec 92 23:38:00 GMT
Distribution: bionet

I realize that this reply is a bit late but our E mail port
was not working properly:  D.G.

----------------Beginning of message----------------------------------
Greetings:

Does anyone have any suggestions as to a good miniprep protocol for the
MC1061/P3 cells?  They are not the most compliant cells when it comes to
minipreps.
Standard alkaline-minis provide degraded DNA.  Promega mini-prep systems
fail completely and yeild NO DNA.  An altered alkaline mini prep protocol
(ala Maniatis) provides DNA but can not be digested.  Does anyone have any
suggestions?

Thanks in advance,

David
haviland@kids.wustl.edu
---------------end of 
message------------------------------------------------------------------


Hi David,
    We have had this problem lately when we had to use another than our
favourite lab E.coli strain (which happens to be DH5alpha).  I think it was to
 generate
hypo methlyatedDNA in a dam- dcm- strain.  Anyway, when Mg++ was added to
 digest the plasmids we hadmajor amounts of degradation.  We solved it by 
being
 very careful the way we handle the bugs during the alkaline minis.  The two 
ml
 cultures were cooled in an ice water bath beforecentrifugation to pellet the 
cells
 (Which is done in the cold room). Everything was doneto keep the whole prep
 as cold as possible during extraction (normally with Dh5 alpha we
 never have to worry about temp).   I think another critical step is the
single phenol:chloroform extraction  after  the K acetate pptte.  
Normally with DH5 alpha we just vortex these for 5-10 sec and spin. 
 But we found if we vortexed the phenol:chloroform extractions from
 the other strain for 1-2 full minutes per tube the degradation problem is 
gone!
 We now do this with all strains we are unfamiliar with.  
Hope this helps.

Dan Gietz
GIETZ@BLDGHSC.LAN1.UMANITOBA.CA
From: pakapke@IASTATE.EDU
Newsgroups: bionet.molbio.methds-reagnts
Subject: electroporation
Date: 13 Dec 92 16:29:34 GMT


     We would like to get away from in vitro packaging our phage DNA and
simply electroporate it into the cell directly.  Anyone in net-land had
experience doing this?  Are there any references published using this
technique to produce a phage library?
From: news@massey.ac.nz (USENET News System)
Subject: Re: colony screening with Amersham ECL (RPN 3000 and 3004)
Organization: Massey University
X-Useragent: Nuntius v1.1
Date: Mon, 21 Dec 92 02:52:01 GMT

In article <1992Dec16.151837@genethon.fr> Christiane Bouchier,
bouchier@genethon.fr writes:
>A week ago I asked whether anyone had ever carried out tests for
screening
>colonies  with Amersham ECL direct system. So far I received no
suggestions
>that could help me overcome the problem we have of detecting peroxydase 
>specific hybridization because of a high background.
>Once again thanks for your help !

 We have had similar problems of background with the ECL system.  We
overcame the problem by including the milk powder (2%) blocking agent in
every step i.e pre-hyb, primary antibody, secondary antibody, and  during
washing of filters. The background was considerably reduced.  Another
trick we found to increase sensitivity was to use filters from other
companies.  Even though they were all marketed as 0.45um nitrocellulose,
we found different (and reproducible) levels of senistivities.
I hope this is of some help.
																								Eamonn.




 *******************************************
 *Eamonn Gormley,  E-mail, E.P.Gormley@massey.ac.nz *
 *******************************************
From: robison1@husc10.harvard.edu (Keith Robison)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: amp gene with internal tga stop mutations wanted
Date: 14 Dec 92 04:26:03 GMT
Distribution: bionet

You might try looking up some of Kurnit's papers on
recombinational screening of libraries.  I found a
reference to a CAT gene, but it is newer and I think
an ampR gene was used previously.  Try using the following
as a start:

	Gene   106:97
		23:343



Good luck!

Keith Robison
Harvard University
Department of Cellular & Developmental Biology
Department of Genetics / HHMI

robison@biosun.harvard.edu
From: DMEIER@mis.mcw.edu
Newsgroups: bionet.molbio.methds-reagnts
Subject: How to empower RNases?!
Date: 11 Dec 92 21:20:49 GMT
Distribution: bionet
Organization: Medical College of Wisconsin (Milwaukee, WI)

Hello All,l
  I'm sure this is a strange request to many of you who have labored 
long and hard to remove pesky RNAses, but I'd like to stabilize or 
increase RNase A and/or RNase T1.  I haven't been adding Mg to these 
incubations... Will Mg or Mn stabilize/increase RNase activity at 37 C
reaction temperatures, and will they stabilize RNAse A during long term
storage at - 20 C in 50% glycerol solutions.
  Thanks much.

  Dan
  DMEIER@MIS.MCW.EDU
From: LCNAVOT@WEIZMANN.WEIZMANN.AC.IL (Nir Navot)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Mobility Shift Assay
Date: 15 Dec 92 14:13:15 GMT
Distribution: bionet
Original-To: METHODS@uk.ac.daresbury

I've been trying to perfect the conditions for running a Mobility Shift Assay.
I'm using this assay to measure the DNA-binding activities of various proteins.
The probe is a 20 mer ds piece of DNA with 4 bases extension at each end and it
 is labled by kinasing. I have been runing the reactions in 4% acrylamide gels
in 0.5xTBE, at 50-100 V at 4 C (cold room). The bands I'm getting are smeared
and smilling (though I'm not).
Could I please have your comments regarding the following points:
1. Presence of protruding ends on the labled oligo
2. Kinasing vers. filling-in for labaling
3. Poly d(I-C) as non-specific competitor vers. sonicated calf-thymus DNA
4. The use of 0.5 x TBE as running buffer, and running conditions in general
5. Your chosen loading buffer
6. Any other tricks?
Thanks in advance!!!
Nir
Newsgroups: bionet.molbio.methds-reagnts