HDTenEightyP/NetNews huggingface dataset:
From: bsh@MED.PITT.EDU (Basavaraju Shankarappa)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: N2 storage of murine cell-lines
Date: 27 Aug 92 13:00:56 GMT
Distribution: bionet

> >I've had problems in successfully freezing macrophage cell cultures so they 
> >can be successfully resuscitated. The problem does seem to lie in the actual freezing. The cells are harvested in the log phase, suspended in a glycerol freezing mixture at five-fold concentration then left overnight (well insulated) in a -70C freeze
> >Can anybody suggest alternative procedures? The alternatives facing me at present are constructing a "freezing box" which will cool at the correct rate in a -70C freezer (feasible) or persuading the department to buy a commercial cell freezer (less f
> >
> >Sean Shaw
> >Microbiology, Reading University
> >skrshaw@sksscsc1.rdg.ac.uk
> Forget the need for commercial freezing machines.
> For many years I have been able to successfully freeze a wide range of
> mammalian cells including mouse and cattle cells, monoclonals etc. with
> excellent retrieval rates (sometimes > than 90%). The cells are simply
> suspended in sterile 90% Foetal Calf serum, 10% DMSO, frozen overnight at
> -70*C in a styrofoam container and then stored in liquid nitrogen (some
> have been down more than 10 years and still revive).  Retrieval is by quick
> thawing in a 37*C water bath until JUST thawed (do not warm to 37) and
> IMMEDIATE resuspension in fresh COLD medium (DO NOT pellet the cells before
> resuspension in the fresh medium).  If the dilution is more than 10 fold
> i.e. the DMSO is reduced to below 1% you do not even need to spin down and
> wash the cells, just plate directly.  If not pellet gently, resuspend
> gently and then plate as per normal.
> 
> I hope that this works for you as it does for me.
> 
> Cheers, Klaus.
> ----------------------------------------------------------------------------
> Klaus Matthaei
> Gene Targeting
> The John Curtin School of Medical Research
> The Australian National University
> E-mail: Klaus.Matthaei@anu.edu.au
> 
> "Don't try and fix it if it ain't broke".
I second Klaus' opinion with great pleasure.  I had actually forgotten
that I had used almost the same procedure which was given to me by
someone I cant remember.  But one thig is for sure.  I had very good results
with this procedure.  In fact we had a LN2 mishap and I was able to 
resuscitate one of the very important hybridoma cell line that was 
frozen with this procedure.  
	I also agree with Klaus' observation that you do not need to
spin the cells to remove DMSO.  I have been given to understand that
it just evaporates in the incubator, if sufficiently diluted.  I 
think it is more of a trauma for the cells to be spun rather than
diluting out the DMSO.  
	One difference from the above protocol I had followed was, after
thawing the vial (approx 1 ml), I added the contents directly to the 
flask that contained about 15 ml of the media.  

So here is another vote for BFS-DMSO
Raj Shankarappa
Univ of Pittsburg
bsh@phobos.med.pitt.edu

flask that conata
Newsgroups: bionet.molbio.methds-reagnts
From: browns@ccu.umanitoba.ca (Stuart Brown)
Subject: Re: PCR buffers
Organization: University of Manitoba, Winnipeg, Manitoba, Canada
Date: Fri, 4 Sep 1992 15:11:41 GMT

In article <loryn.5.0@microbiol.uwa.oz.au> loryn@microbiol.uwa.oz.au (Loryn Sellner) writes:
>We have recently changed our Taq polymerase supplier, and have run into a 
>few problems deciding what reaction buffer to use. The left over 10x buffer 
>from our old Taq source seems to be giving a lot of background non-specific 
>product when used with the new Taq, which we never saw with the old Taq. 
>When we use the new buffer with the new Taq, we get no background, but we 
>also see a ten fold drop in sensitivity. The major differences between the 
>old and new buffer are that the old buffer contained ammonium sulphate as a 
>salt rather than potassium chloride, and also contained gelatin and 2-
>mercaptoethanol, whereas the new buffer does not have these but does 
>have Triton X-100. I would be interested in hearing:
>1) any ideas as to why we are getting these results
>2) anybody else who has encountered problems in using buffers
>3) anybody else who has their own recipe for making PCR buffer
>
>Much appreciated.
>
>
With any new enzyme/buffer combination you should try a range of Mg+
concentrations.

>
>Loryn Sellner
>Department of Microbiology


-- 
Stuart M. Brown                             If you can remain cool when all 
U. of Manitoba, Dept. Plant Science         Around you are in panic,
Winnipeg, Manitoba, CANADA
browns@ccu.umanitoba.ca            Then you surely misunderstand the situation
Organization: Queen's University at Kingston
Date: Wednesday, 2 Sep 1992 08:54:03 EDT
From: <BACKD@QUCDN.QueensU.CA>
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: FinI enzyme info?

Several years ago Szybalski came up with a unique method to cleave
DNA at any site using type II-S restricition endonucleases. Assuming
that FinI can't be found and that the cut must be made at precisely that
site, she may wish to consult the following references:


1. Kim, SC; Podhajski, AJ; Szybalski,W (1988) "Cleaving DNA at any
predetermined site with adapter-primers and class-IIS restriction enzymes"
Science 240:504-506.

2. Podhajski, AJ; Szybalski, W. (1985) "Conversion of the FOKI endonuclease
to a universal restriction enzyme: cleavage of phage M13mp7 DNA at
predetermined sites" Gene 40: 175-182, see erratum in Gene 43: 325

3. Szybalski, W. (1985) "Universal restriction endonucleases: designing
novel cleavage specificities by combining adapter oligodeoxynucleotide and
enzyme moieties" Gene 40: 169-173.

Good luck,

Don Back
Department of Biochemistry
Queen's University
Kingston, Ontario. CANADA.
FR*ATLAS*CICT; arrival Wed, 2 Sep 92 18: 49:25 +0100 action Relayed
From: CAMPBELL@EARN.FRCICT81
Newsgroups: bionet.molbio.methds-reagnts
Subject: URGENT: re-use of hyb solutions
Date: 2 Sep 92 17:49:25 GMT
Distribution: bionet
P1-Message-Id: FR*ATLAS*CICT; 920902183934105-CICT-TLSE
Original-To: methods@UK.AC.DARESBURY
Ua-Content-Id: 920902183934105-

Bonjour,
 
I worked in a lab once where some people froze their used hybridization solution
(containing 32P-labelled probe) and, if the probe was still hot enough,
re-used it for subsequent hybridizations.  They maintained that it worked.
Frankly, I never tried it, so...
 
Does anyone out there have experience with re-using frozen hybridization
solutions containing digoxigenin-labelled oligos?  Does the solution need
to be boiled before use?  Is getting the salmon sperm DNA into "solution"
a problem?  Is probe concentration a problem?  Answers to these questions
and related-experiences greatly appreciated.
 
Merci beaucoup.
 
Malcolm
 
Malcolm M. Campbell
Equipe Lignines
Centre de Physiologie Vegetale
Universite Paul Sabatier
Toulouse
FRANCE
 
CAMPBELL@FRCICT81.BITNET
From: pnh@fcsparc6.ncifcrf.gov (Paul N Hengen)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Partial digestions with ApaL1 - help!!
Date: 1 Sep 92 16:09:22 GMT
Organization: Frederick Cancer Research and Development Center

In article <180321INNc2c@usenet.INS.CWRU.Edu> axa12@po.CWRU.Edu (Ashok Aiyar) writes:
>
>I'm doing partial digestions on a plasmid that has 4 ApaL1
>sites, and can't get the partials to work.  I have tried to
>dilute the enzyme over 2 logs and independently have also tried
>timed digestions.  I seem to get the enzyme cutting at all four
>sites or none of the sites.
>
>Please help!
>
>Ashok

I seem to recall that when digestion of supercoiled DNA occurs,
the first enzyme site is recognized and cutting causes unwinding
that then allows further enzyme activity on the entire relaxed
molecule. It may be worth a try to linearize the DNA by first
cutting with an enzyme that will cut the plasmid only once, and
follow this with timed and/or dilutions of ApaL1.

Paul N. Hengen
National Cancer Institute
Frederick, Maryland  USA
Newsgroups: bionet.molbio.methds-reagnts
From: zxmkr08@mailserv.zdv.uni-tuebingen.de (Cornelius Krasel)
Subject: Re: polysaccharides in DNA extractions
Organization: Comp. Center (ZDV) U of Tuebingen, FRG
Distribution: bionet
Date: Wed, 26 Aug 1992 12:58:33 GMT

In <0095F54C.9C6B8720.236@NTBTOX.NCTR.FDA.GOV> wmelchior@NTBTOX.NCTR.FDA.GOV ("Bill Melchior, NCTR/FDA") writes:

>>I am
>>looking for a way to chemically or enzymatically digest the poly-
>>saccharide without damaging the DNA.  Since DNA is also a 
>>substituted polysaccharide, is this still possible?

>DNA and RNA are not polysaccharides in the usual sense, since they're
>linked through phosphate esters rather directly from sugar to sugar.
>I don't know for sure, but would be surprised if enzymes that digested
>one type of molecule would harm the other.  Trying chemical degradation 
>of polysaccharides without damaging DNA could be riskier.

BioRad offers an enzyme called Gelase for the digestion of Agarose to
purify DNA from Agarose gels. The people in our lab who use it are very 
satisfied with the results. Anyways, I don't know anything about the
substrate specifity of Gelase concerning other carbohydrates.

Cornelius.
--
/* Cornelius Krasel, Department of Physiological Chemistry, U Tuebingen    */ 
/* email: krasel@mailserv.zdv.uni-tuebingen.de (Internet)                  */
/*        krasel@chemie.uni-tuebingen.dbp.de (WIN/X400)                    */
/* "People are DNA's way of making more DNA." (anonymous)                  */
From: wmelchior@NTBTOX.NCTR.FDA.GOV ("Bill Melchior, NCTR/FDA")
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: fin i enzyme info?
Date: 2 Sep 92 11:12:35 GMT
Reply-To: wmelchior@NTDOC.NCTR.FDA.GOV
Distribution: bionet

>A colleauge of mine at Harvard asked me if I would post a request for
>*any* information anyone might have on a restriction enzyme known as Fin I
...
>Failing that, would anyone know the primary reference for this enzyme?
>And, failing that, does anyone know what organism Fin refers to?
____________________________________________________________________________
The Kessler & Manta compendium (Gene 92, 1-248 (1990)) says it's from
_Flavobacterium indologenes_.  The reference is to an unpublished
observation by R. Morgan.  (This is probably the same as the R.D. Morgan
given in the references, who has published several restriction enzyme papers
in Nucleic Acids Research.)  A "$" is used in the Gene paper to indicate
commercially available enzymes, and is NOT present for the Fin I listing.

Sorry.  Would REBASE have more up-to-date information?  I've never used it.

P.S.  In 1990, Kessler & Manta were shown as being with Boehringer Mannheim.
________________________________________________________________________________
The opinions stated are mine, not those of NCTR or its sponsoring organizations.

Bill Melchior                                ||   "You have lawyers the way
National Center for Toxicological Research   ||    other people have mice."
Jefferson, AR  72079                         ||
(501) 543-7206                               ||   -Kenneth Duncan, English
                                             ||    Health & Safety Executive,
WMELCHIOR@NTDOC.NCTR.FDA.GOV                 ||    to US regulators
Organization: University of Maine System
Date: Friday, 4 Sep 1992 13:44:51 EDT
From: <REE700A@MAINE.MAINE.EDU>
Newsgroups: bionet.molbio.methds-reagnts
Subject: Quantizing immobilization - any shortcuts?

   We are attempting to optimize the immobilization of the third
strand in a triple helix assay study (although that is immaterial
to the question at hand).  We are using aminopropyl trimethoxy
silane in a toluene solution and are trying to optimize this step.
  We are studying both solution and vapor phase deposition (wrt
concentration, time and technique) for optimum silane coverage on
lithium niobate, lithium tantalate and quartz.
  Obviously, we can use glutaraldehyde and an enzyme as a fairly
quick test for amino groups on the crystal surface.  OTOH, a
quicker test is desireable.  We have dozens (potentially hundreds)
of 2mm x 2mm x .5mm chips which we want to quantize for available
amino groups.  Any quick, reasonable quantitative analysis would be
appreciated.

  In anticipation of the next steps, a similar quantitative test
for carboxyl groups is also desired.
  Also, a good spacer polymer between the probe and its pendant
amino (artifically included) anchor spot is desired.  Are dummy
(noncomplementary) bases good for this or do they interfere with
affinity? (I am not the biochem person in this project - just the one
with net access).  The probe is complementary to an internal part of
the target DNA and I am concerned about "tight" immobilization
interfering with probe activity.

   Although I say Quantitative, I mean relative...

   Please reply via e-mail to :   Jeff Andle
                                  Biode, Inc. & University of Maine
                                  "Because 2 checks R better than 1"

   REE700A@Maine.maine.edu        DoD #3005
From: tabitha@vms.huji.ac.il
Newsgroups: bionet.molbio.evolution
Subject: archives
Date: 21 Aug 92 14:38:55 GMT
Distribution: world
Organization: The Hebrew University of Jerusalem

can somebody tell me where i can download
the archives of this discussion group?
thank you in advance.
-tabitha
From: Mitch.McGrath@UM.CC.UMICH.EDU
Newsgroups: bionet.molbio.evolution
Subject: unsubscribe
Date: 27 Aug 92 13:40:06 GMT
Distribution: bionet

post
Xref: sparky bionet.molbio.evolution:331 bionet.info-theory:785
From: joe@GENETICS.WASHINGTON.EDU (Joe Felsenstein)
Newsgroups: bionet.molbio.evolution
Subject: Re: genome sizes and number of genes summary
Date: 25 Aug 92 23:23:08 GMT
Distribution: bionet


Tom Schneider, in the midst of his posting on genome sizes writes

> that the genome size is 4673600, while the number of genes is 3237.
> This gives:
> 
>     Rfrequency = log2(4673600/3237) = 10.5 bits per site.
> 

What does Rfrequency do for you?  Is it intended to measure the potential
information content per site?  I would have thought that would be
2*(4673600/3237) = 2888 bits per locus, or 2 bits per site (counting the
four symbols A, C, G, T as equally frequent).

I guess I should have been reading the information theory group.

-----
Joe Felsenstein, Dept. of Genetics, Univ. of Washington, Seattle, WA 98195
 Internet:         joe@genetics.washington.edu     (IP No. 128.95.12.41)
 Bitnet/EARN:      felsenst@uwavm
Xref: sparky bionet.molbio.evolution:329 bionet.info-theory:782
From: toms@fcs260c2.ncifcrf.gov (Tom Schneider)
Newsgroups: bionet.molbio.evolution,bionet.info-theory
Subject: Re: genome sizes and number of genes summary
Keywords: genome sizes number genes summary E.coli Yeast Nematode Drosophila Mouse Human
Date: 25 Aug 92 21:19:29 GMT
Followup-To: bionet.molbio.evolution
Organization: Frederick Cancer Research and Development Center

A personal email to me made me realize I have even better data
for E. coli.  Kenn Rudd estimates from his E. coli sequence database
that the genome size is 4673600, while the number of genes is 3237.
This gives:

    Rfrequency = log2(4673600/3237) = 10.5 bits per site.

This will be published in a forthcoming book:

@inproceedings{Rudd.Schneider1992,
author = "K. E. Rudd
 and T. D. Schneider",
title = "Compilation of {{\em E. coli}} Ribosome Binding Sites",
pages = "?-?",
editor = "Jeffrey Miller",
booktitle = "A short course in bacterial genetics: A laboratory
manual and handbook for {{\em Escherichia coli}} and related bacteria",
publisher = "Cold Spring Harbor Laboratory Press",
address = "Cold Spring Harbor, New York",
year = "1992"}

  Tom Schneider
  National Cancer Institute
  Laboratory of Mathematical Biology
  Frederick, Maryland  21702-1201
  toms@ncifcrf.gov
Xref: sparky bionet.molbio.evolution:328 bionet.info-theory:781
From: toms@fcs260c2.ncifcrf.gov (Tom Schneider)
Newsgroups: bionet.molbio.evolution,bionet.info-theory
Subject: Re: genome sizes and number of genes summary
Keywords: genome sizes number genes summary E.coli Yeast Nematode Drosophila Mouse Human
Date: 25 Aug 92 16:19:26 GMT
Followup-To: bionet.molbio.evolution
Organization: Frederick Cancer Research and Development Center

In article <1992Aug24.161335.730@crc.ac.uk> gwilliam@crc.ac.uk
(Gary Williams x3294) writes:

|The latest values I have for the genome sizes and number of genes
|of species in the various genome programs are:
|
|Species        Genome size       No. of genes
|
|E.coli            4.7 Mbp         2,500 to  5,000

The person who gave you the number 5000 did not document it.  I think
it is a guess and should not be used.  2500 is a better estimate.

|Yeast            14   Mbp         8,000

|> @inproceedings{Fangman1981,
|> author = "W. L. Fangman
|>  and V. A. Zakian",
|> editor = "J. N. Strathern and E.W. Jones and J. R. Broach",
|> title = "MISSING",
|> booktitle = "The Molecular Biology of the Yeast Sacchromyces",
|> publisher = "Cold Spring Harbor Laboratory",
|> pages = "28-28",
|> comment = "size of yeast genome is 14000 kbp +/- 20%",
|> year = "1981"}

Good grief, I don't know where my student got this from!  I have the
book right here and there is nothing on pages 28, the date is 1982, not 1981!
I think he messed up.  Anyway, this is a better reference:

@article{Link1991,
author = "A. J. Link
 and M. V. Olson",
title = "Physical Map of the {{\em Saccharomyces cerevisiae}}
Genome at 110-Kilobase Resolution",
journal = "Genetics",
volume = "127",
pages = "681-698",
year = "1991"}

Assuming that rDNA takes 2 to 3 x 10^6 bp, we calculate from the data
in that paper:  1.50E+07 +/-  5.13E+05 bp.  From this estimate and
assuming the frequency of genes is that of orfs found by Oliver et al
(Nature 357: 38 (1992)) is 182/315357, I find an estimate for
the number of genes as 8651 +/- 296.

Of course, all these numbers are important and useful for doing
an information theory analysis.

  Tom Schneider
  National Cancer Institute
  Laboratory of Mathematical Biology
  Frederick, Maryland  21702-1201
  toms@ncifcrf.gov
From: ZWIEB%JASON.DECNET@relay.the.net (Christian Zwieb)
Newsgroups: bionet.molbio.evolution
Subject: monster-bacterium
Date: 2 Sep 92 21:59:42 GMT
Distribution: bionet

I am looking for information on the nature of a large bacterium found in the
gut of fish. Can somebody help? Thanks! -Chris

____________________________________________________________________ 
Christian Zwieb, Department of Molecular Biology
The University of Texas Health Science Center at Tyler
Highway 271 N, P.O. Box 2003 Tyler,TX 75710, U.S.A.      
phone:  (903) 877-7689, (903) 877-7676, fax: (903) 877-7558
e-mail: zwieb%jason.decnet@relay.the.net (internet) 
access to the signal recognition particle database:
ftp 129.117.100.7 (name: anonymous)
____________________________________________________________________
From: danj@WELCHGATE.WELCH.JHU.EDU (Dan Jacobson x-8453)
Newsgroups: bionet.molbio.embldatabank
Subject: Re: How to retrieve sequences from EMBL and Genbank
Date: 2 Sep 92 14:44:51 GMT
Distribution: bionet

J.A. Lenstra and J.J. Batenburg write:

>(....)
>Could you advise us on how to retrieve the 
>sequence of the gene for rat fatty acid synthase from the data base?
>Thank you very much in advance.


I have sent  the 4 sequences in Genbank which pertain to rat fatty 
acid synthase to them.  There are several ways that you can
access this data.

You can use Gopher to retrieve them from fly.bio.indiana.edu (port 70)
for genbank and bioftp.unibas.ch (port 70) for EMBL (if
you don't know what gopher is let me know and I`ll send you some information).
You can also telnet to genbank.bio.net and login as genbank, password is 4nigms
and use the online IRX system to search for them (unfortunately this system
will end at the end of this month).  You can also use the
mailserver search and retrieval at NCBI  or EMBL (again I'll send you info if
you haven't heard about this).  

Best of luck,

Dan Jacobson
From: danj@WELCHGATE.WELCH.JHU.EDU (Dan Jacobson x-8453)
Newsgroups: bionet.molbio.embldatabank
Subject: Sequence retrieval
Date: 2 Sep 92 16:23:13 GMT
Distribution: bionet


Hmm, seems that I forgot to include the PIR mailserver in
my list of ways to access sequences.  The following search
would retrieve the desired sequences from the PIR mailserver.

USE BASES EMBL+EMBLSUP+GBNEW
QUERY
TITLE FATTY ACID SYNTHASE
SPECIES RAT
GET ALL
END QUERY



Kudos go to John Garavelli for jogging my (failing) memory cells.



Dan Jacobson
From: dddzeij@nl.ruu.cc (van der Zeijst)
Newsgroups: bionet.molbio.embldatabank
Subject: no subject (file transmission)
Date: 2 Sep 92 13:07:56 GMT
Distribution: bionet
Original-To: embl-db@uk.ac.daresbury
X-Mailer: ELM [version 2.3 PL11]

Dear colleagues,

We consulted accession number M84761, referred to by Amy et al. 
(PNAS 89, 1992, 1105-1108), who describe the sequence of the gene of 
rat fatty acid synthase. 
However, this number belongs to entry RNTHYBS, dealing with another gene 
(although Amy et al. are cited!). Could you advise us on how to retrieve the 
sequence of the gene for rat fatty acid synthase from the data base?
Thank you very much in advance.

Sincerely yours,

Dr. J.A. Lenstra and Dr. J.J. Batenburg
Newsgroups: bionet.molbio.embldatabank
Subject: Renewal of EMBL CD-ROM/tape subscriptions
From: stoehr@embl-heidelberg.de
Date: 3 Sep 92 11:06:55 +0100
Organization: EMBL, European Molecular Biology Laboratory

Subscribers to EMBL CD-ROM's and magnetic tapes are reminded that September
1992 marks the beginning of a new subscription period. To renew subscriptions
please return the subscription forms which were part of our May mail-shot.
New forms can be sent via e-mail, post or fax on request or can be retrieved
from the EMBL mail or FTP servers.
1- E-mail file server: send e-mail to NetServ@EMBL-Heidelberg.DE containing
                       the line:
                       GET DOC:ORDERFORM.TXT

2- Anonymous FTP     : connect using FTP to FTP.EMBL-Heidelberg.DE and get
                       the (ascii) file in /pub/doc/orderform.txt

Regards,
Peter Stoehr
EMBL Data Library, Postfach 10.2209, 6900 Heidelberg, Germany.
Tel: +49 6221 387258  E-mail: DataLib@EMBL-Heidelberg.DE  Fax: +49 6221 387519