HDTenEightyP/NetNews huggingface dataset:
From: bl275@cleveland.Freenet.Edu (Dan Diaz)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: ? How to destroy DNA chemically (to reuse DNA columns)
Date: 23 Dec 1992 05:41:42 GMT
Organization: Case Western Reserve University, Cleveland, OH (USA)
Reply-To: bl275@cleveland.Freenet.Edu (Dan Diaz)
NNTP-Posting-Host: hela.ins.cwru.edu


In a previous article, djt2@po.CWRU.Edu (Dennis J. Templeton) says:

>Well, the old cheapskate (me) has broken down and bought a bunch of
>silica-based columns for DNA column purification (plasmid).  We went with
>those from Machery Nagel (Nucleobond) bought in the US from The Nest Group.
>These are similar to the Qiagen columns, but the Nest Technical support was
>more informative and they were a little cheaper.

I am confused.  Qiagen columns are DEAE anion exchangers and yet you say
they are similar to a silica column?

Second point: sounds like you are wasting time and effort and taking a
potentially hazardous (in time and money) risk in trying to re-use columns.
Qiagen columns are not *that* expensive.  Spend the extra money and sleep
well at night.  Sure as hell beats troubleshooting.
-- 
Dizzy Dan         ddiaz@cwru.bitnet      bl275@cleveland.freenet.edu
Department of Molecular Genetics - Albert Einstein College of Medicine
"My opinions are my own, and they should be yours as well"
Newsgroups: bionet.molbio.methds-reagnts
From: gilbertd@chipmunk.bio.indiana.edu (Don Gilbert)
Subject: Re: MEDLINE
Organization: Biology, Indiana University - Bloomington
Date: Sun, 27 Dec 1992 13:54:25 GMT


Articles that have been posted to bionet newsgroups in the
past can be found easily using Internet Gopher to the IUBio Archive
(located at ftp.bio.indiana.edu).   In the Network-News/ folder
there is an option to search all of the archived news articles.
I recently found the Medline-thru-Internet information by searching
there on [medline grateful med internet].  Note, you can find prior
articles on methods & reagents here also.

Here is a summary of what you can do to find this info:

  gopher ftp.bio.indiana.edu

                    Root gopher server: ftp.bio.indiana.edu
      1.  About-IUBio-Archive  [ 7Dec92, 24kb].
	  ...
 -->  9.  Network-News/

                                  Network-News
      1.  About Netnews  [13Aug92, 2kb].
 -->  2.  Bionet News at IUBio archive <?>
      3.  Bionet News via WAIS at Net.Bio.Net <?>
      4.  bionet/
          ...

          Bionet News at IUBio archive: medline grateful med internet
      1.  kristoff@G Re: Final 1991 mailing list.
 -->  2.  kleinschmi Re: Medline via Internet & Grateful Med.
      3.  kleinschmi Re: More on Medline and Grateful Med.
      4.  donp@niaid Re: Re: Medline and Internet - any progress?.
	  ...

-- Don
[ If you need help with Internet Gopher, try anonymous ftp to boombox.micro.
umn.edu, in folder pub/gopher/, or to ftp.bio.indiana.edu, in folder
util/gopher/ .  Look for the gopher FAQ document.]
-- 
Don Gilbert                                     gilbert@bio.indiana.edu
biocomputing office, biology dept., indiana univ., bloomington, in 47405
From: djt2@po.CWRU.Edu (Dennis J. Templeton)
Newsgroups: bionet.molbio.methds-reagnts
Subject: ? How to destroy DNA chemically (to reuse DNA columns)
Date: 22 Dec 1992 16:17:17 GMT
Organization: Case Western Reserve University, Cleveland, OH (USA)
Reply-To: djt2@po.CWRU.Edu (Dennis J. Templeton)
NNTP-Posting-Host: thor.ins.cwru.edu


Well, the old cheapskate (me) has broken down and bought a bunch of
silica-based columns for DNA column purification (plasmid).  We went with
those from Machery Nagel (Nucleobond) bought in the US from The Nest Group.
These are similar to the Qiagen columns, but the Nest Technical support was
more informative and they were a little cheaper.

They work fine, as many of you have attested previously.

They are expensive, though, and I'm toying with how to re-use them.  The
critical element is to avoid pH above 9 for long periods (in minutes, I'm
told). The important aspects would be to 1) retain binding ability and 2)
remove DNA that might contribute to the next prep.

We tried washing the column with DNAse solution (200 ug/ml in Mg++
containing buffer, RT for 60 minutes) and re-stripping.  When we
re-stripped again, though, we recovered plasmid "contaminant" that amounted
to about 10e-5 of the original amount, based on amp-containing transfection
units.  I think this is too much to allow re-use.

I'm thinking now about *chemical* methods of degrading/destroying the DNA
bound to the column.  1M NaOH is out (that destroys the silica column) and
I'm not too keen on 4 M HCl for the same reason.  I guess I'm leaning
towards an alkylation agent, maybe Acetic anhydride, though I really don't
have a good feel for what might work best. Maybe an oxidative reagent, like
peracetic acid.

So... any chemists out there?  What should we try that works near neutral
pH in aqueous or anhydrous conditions that should destroy the biological
activity of the residual DNA, that is safe and relatively cheap?  If you
have reasonable suggestions, I'll try it out and post a followup.

dennis
Organization: University of Illinois at Chicago
Date: Wednesday, 30 Dec 1992 13:40:03 CST
From: Lixin Zhou <U09723@uicvm.uic.edu>
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Plasmid DNA Extraction

In article <92364.221112U09723@uicvm.uic.edu>, Lixin Zhou <U09723@uicvm.uic.edu>
says:
>
>I got troubles in extracting plasmid DNA from Streptococcus
>pneumoniae (Gram-positive).  The plasmid is about 15 kb.
>Pneumococcal chromosomal DNA and plasmid DNA are AT rich (about
>62%).  The method I used is a modified alkali lysis protocol.
>Solution II contains 1% SDS and 0.175 N NaOH instead of 0.2 N NaOH.
>Solution III contains 50 mM Tris.Cl-pH8.0 in addition to 0.1 M
>NaAc-pH4.8.
> ....

I have found a mistake in the modified protocol.  0.1 M NaAc-pH4.8
in Solution III should be 3 M.  I misunderstood the modified protocol
which was not mine.

I appreciate the suggestions appearing in Bionet and delivered in my
personal mailbox.

With apologies,

Lixin Zhou
Laboratory for Molecular Biology, University of Illinois at Chicago
From: parkhillj@vax1.bham.ac.uk
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: T-tracking program ?
Date: 22 Dec 92 10:17:09 GMT
Organization: University of Birmingham
NNTP-Posting-Host: vax3.bham.ac.uk

In article <1992Dec21.083510.13693@abo.fi>, JANIEMI@FINABO.ABO.FI (Jarmo Niemi TUY) writes:

> I am looking for a program, that helps one to identify the
> position of a clone in a sequence by running only one
> sequencing reaction (so-called T-tracking). Some sources,
> like Deininger's review (Anal. Biochem 135(1983)247-263) and
> Amersham's sequencing leaflet state that this can be done
> by computer, but I haven't so far found any reference to
> a program that would do it. 
> 
> Otherwise I use the GCG fragment assebly programs, but
> haven't been able to figure out how to map T-tracks 
> by GCG. Any ideas would be appreciated.
 
 Jarmo,

	I don't know  of any specific programs to do this, but it
can be done in GCG by writing out your sequnce using "T" and "V"
(where V = A or C or G), and then using this sequence in either
GAP or BESTFIT.
	The hardest part is estimating the number of gaps between
the T's

hope this helps

Julian Parkhill.
From: vjongene@isrec-sun1.unil.ch (Victor Jongeneel)
Subject: Re: T-tracking program ?
Organization: University of Lausanne CH (Switzerland)
X-Newsreader: TIN [version 1.1 PL8]
Date: Tue, 22 Dec 1992 12:58:22 GMT

Jarmo Niemi TUY (JANIEMI@FINABO.ABO.FI) wrote:

: I am looking for a program, that helps one to identify the
: position of a clone in a sequence by running only one
: sequencing reaction (so-called T-tracking). Some sources,
: like Deininger's review (Anal. Biochem 135(1983)247-263) and
: Amersham's sequencing leaflet state that this can be done
: by computer, but I haven't so far found any reference to
: a program that would do it. 

Jarmo,

A T-track identification method has been published by Golovanov and
Nedospasov in CABIOS 2:297, 1986.  It has the great advantage of _not_
making you guess the gap between groups of T's, which would be the
greatest source of errors if you used "classical" sequence analysis
programs such as those found in GCG.  The principle is that it
generates a numerical code for a T-track which is based on the
occurrences of T singlets and multiplets in a nucleotide
sequence.  The code obtained from a gel can then be compared to a
"library" of codes generated from the reference sequence.

I do not have the program source code available, but can direct you to
Sergei Nedospasov if you want it.  My guess is that it would be a
trivial programming job anyway, once the technique is understood.

Good luck!


--------------------------------------------------------------------
C. Victor JONGENEEL                       vjongene@isrecmail.unil.ch
Ludwig Institute for Cancer Research
Chemin des Boveresses 155                       FAX: +41-21-653-4474
CH-1066 EPALINGES				Tel: +41-21-316-5994
Switzerland
--------------------------------------------------------------------
From: DMEIER@mis.mcw.edu
Newsgroups: bionet.molbio.methds-reagnts
Subject: Commercial peptide sources
Date: 21 Dec 92 20:12:43 GMT
Distribution: bionet
Organization: Medical College of Wisconsin (Milwaukee, WI)

Hello All,
  At least two companies advertise, in the back of Science, custom 
synthesis of peptides which will be shipped to the customer in 10 days. 
  Does anyone have experiences (good or bad) with these companies? 
Are these companies reliable (Research Genetics, Bio-synthesis)?  Can 
someone please recommend a company they have had personal experience 
with or know someone who has had a a good experience.
  These companies are appealing since they charge $30.00/residue which 
seems reasonable.
  Any replies gratefully received.

Dan Meier
DMEIER@MIS.MCW.EDU
Newsgroups: bionet.molbio.methds-reagnts
From: JANIEMI@FINABO.ABO.FI (Jarmo Niemi TUY)
Subject: T-tracking program ?
Organization: Abo Akademi University, Finland
Date: Mon, 21 Dec 1992 08:35:10 GMT
X-News-Reader: VMS NEWS 1.24

I repost the following from bionet.software, as I am not
getting any response there...

I am looking for a program, that helps one to identify the
position of a clone in a sequence by running only one
sequencing reaction (so-called T-tracking). Some sources,
like Deininger's review (Anal. Biochem 135(1983)247-263) and
Amersham's sequencing leaflet state that this can be done
by computer, but I haven't so far found any reference to
a program that would do it. 

Otherwise I use the GCG fragment assebly programs, but
haven't been able to figure out how to map T-tracks 
by GCG. Any ideas would be appreciated.

Jarmo Niemi   Biochemistry, University of Turku
              SF-20500 Turku
              ++358-21-6335758
              janiemi@finabo.abo.fi

"You may quote me"
	-this sentence
		(quoted from Douglas R. Hofstadter:Metamagical themas)
			{therein reprinted from Scientific American,
			January, 1981}
				[and probably published even before]


Newsgroups: bionet.molbio.methds-reagnts
From: gat44@iastate.edu (Gabriel Trueba)
Subject: MEDLINE
Organization: Iowa State University, Ames, IA
Date: Sat, 26 Dec 1992 19:42:05 GMT

 A few months ago there was a posting indicating how to obtain
access to MEDLINE for free. I would appreciate if somebody can
send this information.

G. Trueba
gat44@iastate.edu
|
From: u6069416@ucsvc.ucs.unimelb.edu.au
Newsgroups: bionet.molbio.methds-reagnts
Subject: Request for tobacco seeds information
Date: 22 Dec 92 13:43:12 +1100
Organization: The University of Melbourne

I repost the following from bionet.plants, as I got only two answers, both of
them very useful, but I hope somebody else could have more information.
Does anyone know where could I find information, mostly pictures and diagrams,
about the anatomy and citology of tobacco seeds? I am going to test GUS 
expression directed by a barley seed-specific promoter in transgenic tobacco
plants and I would like knowing more about this organ before starting. My main
concern is being able to recognize in microscope preparations the diferent
tissues of tobacco seeds during several developmental stages.
Thank you for your answers.

Joaquin Royo
School of Botany
University of Melbourne
Australia
E-mail:
         u6069416@ucsvc.ucs.unimelb.edu.au
From: eric@bcserv.wustl.edu (Hugo (Eric))
Newsgroups: bionet.molbio.methds-reagnts
Subject: Sequence of the mouse DHFR gene
Date: 2 Jan 93 18:27:14 GMT
Organization: Washington University in Saint Louis, MO USA
NNTP-Posting-Host: bcserv.wustl.edu
Keywords: Mouse DHFR nucleotide seq

Dear Bionetters,
     I am seeking the nucleotide sequence for the cDNA encoding
the mouse dihydrofolate reductase gene product.  A Genbank search
turned up only promotor regions of the mouse gene.  I am particularly
interested in the sequence of the cDNA described in:
Murray, et al.  1983.  Mol. Cell Bio. 3(1):32-43.
The plasmids discussed in this paper are:
pDHFR26 and their construct pLTRdhfr26.  Any assistance would be greatly
appreciated.  Please email replies to help reduce network bandwidth.
Thank You!                                   Eric ;-)
-- 
  Eric R. Hugo, Postdoctoral Research Associate |eric@bcserv.wustl.edu
  Dept. of Biochemistry and Molecular Biophysics|                
  Washington University School of Medicine      |               
  Box 8231, St. Louis, MO 63110_________________| (314) 362-3342
Newsgroups: bionet.molbio.methds-reagnts
From: ashok@biochemistry.cwru.edu (Ashok Aiyar)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: MEDLINE
Date: Sun, 27 Dec 1992 00:47:22 GMT
Organization: CWRU School of Medicine
NNTP-Posting-Host: axa12-slip.dialin.cwru.edu

In article <BzvtE6.D1F@news.iastate.edu> 
	gat44@iastate.edu (Gabriel Trueba) writes:


> A few months ago there was a posting indicating how to obtain
>access to MEDLINE for free. I would appreciate if somebody can
>send this information.

As far as I know, MedLine access is *not* free.  You can subscribe to 
MedLine via a CD-ROM service, or use MedLines over the Internet using 
Grateful Med, but you pay for the service nonetheless.

I had posted a message few months back stating that Grateful Med Ver 6.0 
(the latest IBM PC version) worked well over TCP/IP connections, and in 
particular worked over SLIP connections to the Internet as well.

If you want to use Grateful Med over a SLIP connection on a PC, you must use 
the EtherSLIP packet driver.  Grateful Med 6.0 uses the TCP/IP stack from 
NCSA Telnet ver 0.94, and this particular version does not talk well to 
other SLIP packet drivers such as SLIP8250 or SLP16550.

Ashok

----------------------------------------------------------------------
                              Ashok Aiyar 
                      Department of Biochemistry
                       CWRU School of Medicine
----------------------------------------------------------------------
Newsgroups: bionet.molbio.methds-reagnts
From: vjongene@isrec-sun1.unil.ch (Victor Jongeneel)
Subject: Re: supercoiled DNA migration in agrose ?
Organization: University of Lausanne CH (Switzerland)
X-Newsreader: TIN [version 1.1 PL8]
Date: Mon, 28 Dec 1992 06:57:42 GMT

chai_z@wehi.edu.au wrote:

: Hi,

: 	Several batches of M13 RF DNA of mine have been cut by EcoRI or SmaI,
: etc. and then electrophoresized in agrose gel. Uncut DNA (supercoiled) of some
: batches migrated faster than the cut one (linear), while the others slower.
: Most likely, some batches of my DNA were nicked (open circular). Could any one
: tell me the correct agrose pattern of supercoiled, open circular and linear DNA?
: or tell me where to refer to?

: Thanks in advance.

: Zhonglin Chai 


In most agarose gels (but _not_ always), supercoiled circular DNA
migrates fastest, followed by linear and nicked circles.  Linear does
not always separate from supercoiled, nicked circles usually do.  This
does of course not exclude the presence of miniphage (see other
response in thread).  The best way to find out is to start
with freshly purified (CsCl/EtBr) supercoiled DNA and treat it so as
to make linears and nicked circles.

Cheers,


--------------------------------------------------------------------
C. Victor JONGENEEL                       vjongene@isrecmail.unil.ch
Ludwig Institute for Cancer Research
Chemin des Boveresses 155                       FAX: +41-21-653-4474
CH-1066 EPALINGES				Tel: +41-21-316-5994
Switzerland
--------------------------------------------------------------------
From: BIOSEE@UKCC.UKY.EDU (Stephanie Edelmann)
Newsgroups: bionet.molbio.methds-reagnts
Subject: labelling dog muscle cells (primary culture)
Date: 23 Dec 92 16:06:17 GMT
Organization: University of Kentucky

As an infrequent reader of this group, I don't know if this question is
appropriate in this context.  If not, I'm sorry and where should I post
it?
 
Here's my problem.  I am supposed to label a primary dog muscle cell
culture so that the cells can be recognized when they are reinjected
in the same dog.
 
1. Is it feasable (sp?) to use a marker-plasmid to do that or
are the cells going to be senescent by the time I have transfected
and selected them?  (I have never worked with primary cell culture)
 
2. Should I go to a retroviral vector?  They are supposed to be pretty
selective in their host.  What vectors could be used in a dog?
 
3. Where could I find information of this kind?  Not only the above
questions, but things like "general" transfection procedures etc.
 
I realize that these questions are very general and there's probably
some easy place to find all this.  So,please excuse my ignorance. I am
new to this.
Please e-mail replies since I don't have access to the net very often.
 
Thanks, Stephanie
From: LCNAVOT@WEIZMANN.WEIZMANN.AC.IL (Nir Navot)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Mobility Shift
Date: 21 Dec 92 14:51:43 GMT
Distribution: bionet
Original-To: METHODS@uk.ac.daresbury

Many thanks to all you folks who answered my question on Mobility Shifts!!!

Nir
    Navot.
From: djt2@po.CWRU.Edu (Dennis J. Templeton)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: ? How to destroy DNA chemically (to reuse DNA columns)
Date: 23 Dec 1992 19:34:52 GMT
Organization: Case Western Reserve University, Cleveland, OH (USA)
Reply-To: djt2@po.CWRU.Edu (Dennis J. Templeton)
NNTP-Posting-Host: thor.ins.cwru.edu


In a previous article, bl275@cleveland.Freenet.Edu (Dan Diaz) says:

>
>In a previous article, djt2@po.CWRU.Edu (Dennis J. Templeton) says:
>
>>Well, the old cheapskate (me) has broken down and bought a bunch of
>>silica-based columns for DNA column purification (plasmid).  We went with
>>those from Machery Nagel (Nucleobond) bought in the US from The Nest Group.
>>These are similar to the Qiagen columns, but the Nest Technical support was
>>more informative and they were a little cheaper.
>
>I am confused.  Qiagen columns are DEAE anion exchangers and yet you say
>they are similar to a silica column?

Both columns are anion exchangers (subtly different) on a silica matrix.

>
>Second point: sounds like you are wasting time and effort and taking a
>potentially hazardous (in time and money) risk in trying to re-use columns.
>Qiagen columns are not *that* expensive.  Spend the extra money and sleep
>well at night.  Sure as hell beats troubleshooting.
>

You have a point, but our use here will consume ca $6000 per year.  Here's
a comparison: would you throw away 10 FPLC columns a year at $600 each to
sleep better at night?  maybe you or some would.  This is a variation of
the "kit mentality" theme discussed here several times.  I think there are
valid arguments on both sides of the discussion.
Newsgroups: bionet.molbio.methds-reagnts
From: wjb1@ellis.uchicago.edu (William J. Buikema)
Subject: Lorist 6 vector information
Reply-To: wjb1@midway.uchicago.edu
Organization: University of Chicago Computing Organizations
Date: Wed, 30 Dec 1992 05:02:31 GMT

I'm trying to find information about a cosmid cloning vector called Lorist 6.
I am aware of a paper describing Lorist 2 a few years back in Gene, but now 
I need a reference or map of this later version.  Any help will be appreciated.


William J. Buikema              internet: wjb1@midway.uchicago.edu
University of Chicago               or    w-buikema@uchicago.edu
Dept. of Molecular Genetics &       or    hetman@uchicago.edu 
    Cell Biology, CLSC 313
920 E. 58th St.
Chicago, IL  60637
From: pakapke@IASTATE.EDU
Newsgroups: bionet.molbio.methds-reagnts
Subject: (none)
Date: 21 Dec 92 22:48:28 GMT

Does anyone have the Sau 3A methylase gene cloned and expressed in a gram 
negative host?  We are looking for a source that might have that gene
expressed in such a host.  We are having a restriction barrier in 
our host system and feel it is a Sau 3A like restriction barrier.

Also, does anyone know of a E coli cloning vector that does not have 
any Dra I sites.  All the puc vectors have Dra I sites in the Amp gene
as does the pBC vector in the chloramphenicol gene.

Thanks.
Newsgroups: bionet.molbio.genbank
From: robison1@husc10.harvard.edu (Keith Robison)
Newsgroups: bionet.molbio.genbank
Subject: EMBL <> GenBank
Date: 22 Dec 92 19:21:43 GMT
Organization: Harvard University Science Center


	I have noticed a small but significant sprinkling of sequences
which are in the EMBL database but not in GenBank.  The most recent
case concerns a sequence which was updated significantly in August,
yet the revised sequence has not yet migrated yet.

1)  Why is this happening.  What happened to the goal of keeping the
    sequence content of EMBL and GenBank equal.

2)  Would the folks at NCBI consider generating a "Translated EMBL"
    for their Sequence and BLAST servers so that derived translations
    (temporarily?) unique to EMBL are accessible?


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

robison@biosun.harvard.edu
From: GenBank-Updates@net.bio.net
Newsgroups: bionet.molbio.genbank.updates
Subject: Human HepG2 3'-directed MboI cDNA, clone tw1-44
Date: 29 Dec 92 00:44:23 GMT
Distribution: bionet
Checksum: 65061 1

LOCUS       HUM00TW144     67 bp ds-DNA             PRI       03-DEC-1992
DEFINITION  Human HepG2 3'-directed MboI cDNA, clone tw1-44
ACCESSION   D12419
KEYWORDS    expressed sequence tag.
SOURCE      Homo sapiens Male Liver cDNA to mRNA.
  ORGANISM  Homo sapiens
            Eukaryota; Animalia; Chordata; Vertebrata; Mammalia; Theria;
            Eutheria; Primates; Haplorhini; Catarrhini; Hominidae.
REFERENCE   1  (bases 1 to 67)
  AUTHORS   Okubo,K., Hori,N., Matoba,R., Niiyama,T., Fukushima,A., Kojima,Y.
            and Matsubara,K.
  TITLE     Large scale cDNA sequencing for analysis of quantitative aspects of
            gene expression
  JOURNAL   Nature Genet. 2, 173-179 (1992)
  STANDARD  full automatic
BASE COUNT       15 a     16 c     22 g     14 t
ORIGIN      
        1 gatcgggccc gggggctgag cctgggaacc ccaccatttg ttaatgaaaa atgagttttg
       61 gcagcgc
//