HDTenEightyP/NetNews huggingface dataset:
From: afc@gnv.ifas.ufl.edu
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: hybridization problems
Date: 13 Oct 92 10:36:14 -0500
Distribution: bionet

In article <1992Oct12.090837.24582@gserv1.dl.ac.uk>, ED@micro.uct.ac.za (Ed Rybicki) writes:
>> From:       s882076@yallara.cs.rmit.OZ.AU (Sean Davidson)
>> Subject:    Re: hybridization problems
>> Date:       10 Oct 92 03:11:10 GMT
> 
>> Further to this discussion about different membranes, I recently began using
>> the Digoxigenin-based system for Northern blotting. Initial attempts used
>> Hybond N+, which is recommended for this procedure, but signal was always
>> virtually undetectable amongst high background. When I began using
>> Zetaprobe (Biorad) instead, results were (are) great.
>>   So do you think this may have just been the batch of Hybond, or why are
>> the results so much different?
> 
> I have seen a lab in the US to whom I had extolled the virtues of DIG,
> fail miserably in many attempts to get Northerns to work in an environment
> where 32P blots had been working well.  They also used to get "negative
> bands" where the RNA should have been.  The local reps were of the
> opinion that they should try the specialist Northern kit with RNAse-free
> everything.  I then came home to find people gaily Northerning with plant
> viral RNA and DNA probes with DIG, with excellent results - and they were
> using Hybond-N, whereas my American colleagues were using AN other brand.
> The answer is, who knows?  Probably one problem is the presence of RNAses
> in the various blocking agents, etc, used with DIG - but then why does our
> Genius kit work with our RNA?  Do we have a different batch of Boehringer
> blocking agent?  Ours is still working, with chemiluminescence, and dye.
> Maybe it'll stop, and we won't know why either...!  Will share protocols
> if anyone is interested.

Our experience was similar to the anonymous US lab.  We compared the
Genius kit to Amersham's chemoluminescence kit, BRL's strepavidin/alk
phos, and P-32.  We always had more background and lower signals with
the Genius kit.  I don't know if it was filters or what... we just
used one of the other that worked.

Andrew Cockburn
USDA

>  ____________________________________________________________________
> | Ed Rybicki, PhD             |    "Now you've got the hang of it    |
> | (ed@micro.uct.ac.za)        | There's nothing you can't do with it |
> | Dept Microbiology           |        If you're very into it        |
> | University of Cape Town     |         You can't go wrong...."      |
> | Private Bag, Rondebosch     |                                      |
> | 7700, South Africa          |               -Mad John              |
> | fax: 27-21-650 4023         | (Ogden's Nut Gone Flake, Small Faces)|
>  --------------------------------------------------------------------
From: PHILLIPSA@LARS.AFRC.AC.UK (Andy Phillips)
Newsgroups: bionet.molbio.methds-reagnts
Subject: UK source for Savant gel pump
Date: 14 Oct 92 09:19:00 GMT
Distribution: bionet
Original-To: METHODS@UK.AC.DARESBURY.DLGM

We need to replace our ancient and unreliable gel
drying pump (an Aquavac) soon. The consensus on the
network appears to be that the Savant gel pump is
the best acid-resistant pump on the market. Does anyone
have an address/phone/FAX for a supplier in the UK?

Thanks
Andy Phillips (PHILLIPSA@LARS.AFRC.AC.UK)
Long Ashton Research Station
From: steve@rbiffm.informatik.uni-frankfurt.de (Stefan Zickenheiner)
Newsgroups: bionet.molbio.methds-reagnts
Subject: searching media for thermoproteus tenax
Date: 8 Oct 92 11:06:57 GMT
Organization: J. W. Goethe-Universitaet Frankfurt/Main, Germany


Hello out there,

I am looking - for a friend of mine not familiar in using Unix-stations -
for a specific media for thermoproteus tenax, a sulphur-reducing bacterium
which does not grow the way we want to (in several attempts).

We are referring to a copy of an old article (1981), but the media described
there is not of great use, in fact, we can't even create a RICH media in
which our bacterium feels well (it grows a bit, though).

I don't think we are working essential wrong (we know it has to be tempered
and is growing under absence of oxygen, of course!) it is "just" the wrong
media... (hard work to find the right one, I know!)

Are there any good references, or does anybody work in that direction??

Thanks a lot, Steve.

Please feel free to e-mail me on Internet:

steve@ti.informatik.uni-frankfurt.de
From: cytogen@chmeds.ac.nz (Martin Kennedy)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: RT-PCR
Date: 11 Oct 92 22:29:02 GMT
Distribution: bionet

In article <9210090425.AA07044@cscgpo>, Klaus.Matthaei@anu.edu.au writes:
> G'Day Netters
> 
> I have some questions regarding PCR from RNA.  The standard method is to
> use Reverse Transcriptase and oligi-DT to synthesise cDNA from total RNA
> followed by standard PCR.  Another method is to use the dowmnstream primer
> for the cDNA synthesis followed by PCR.  More recently Amersham has brought
> out a taq polymerase called Tet-Z which has both RT and polymerase activity
> (indeed Amplitaq from cetus does this also).  The reverse transcription and
> the PCR is therefore done in the same tube in the same set of cycling
> reactions, the first one being a 15-30 minute RT step followed by standard
> PCR.  We have also tried these methods (when I say we, read 1 honours

 (STUFF DELETED)
> 
> Any help will be appreciated.
> 
> Cheers, Klaus
> --------------------------------------------------------------------------
> Klaus Matthaei



Klaus:

If you are just looking for RT-PCR methods that work rather than direct
experiences with Tet-Z, I do quite a lot of RT-PCR for isolating probes
and for anchored-PCR and I use the following:


First strand cDNA synthesis:
		
		5ug total RNA (or 1-2ug polyA if available)
		1ul RNasin 
		H2O up to 13ul final vol

Incubate 65oC for 5 min, then -> ice		

Add:		1ul of oligo dT (or GSP) @ 0.5ug/ul
		1ul RNasin
		4ul 5 X buffer + dNTPs
		1ul Reverse transcriptase

I use total RNA (poly-A not necessary) isolated by phenol/SDS extraction; 
RT primed with either oligo-dT or a gene specific primer, with LifeTech-BRL 
Superscript and the buffer supplied with this enzyme(but you must add in all
four dNTPs).  

Incubate 42oC for at least 1 hour.

Add water up to 100ul final vol, and heat at 70oC for 10 minutes.

This is ready to use in PCR - just take 5ul into a 50ul PCR rx, and go for it. 
No need to remove the diluted primer.
		
If you want to do anchored PCR or clean up the cDNA further, then I do 3 washes
through a Centricon-30 spin dialysis cartridge, with 1ml H2O each time.

Hope this is useful,

Cheers,
Martin
From: RGB12955%USA.decnet@USAV01.GLAXO.COM ("USA::RGB12955")
Newsgroups: bionet.molbio.methds-reagnts
Subject: re: PCR max. lenght
Date: 7 Oct 92 19:44:00 GMT
Distribution: bionet

In their recent (Sept. 92) publication "NEB Transcript" NEB claims to 
have extended primers over 13kb.  They don't say whether they managed to 
isolate a fragment from that extension.

Rich Buckholz "no association with NEB"
From: KNECHT@UCONNVM.UCONN.EDU
Newsgroups: bionet.molbio.methds-reagnts
Subject: PCR cyclers
Date: 14 Oct 92 19:32:38 GMT
Distribution: bionet

Hi- I am in the market for a PCR cycler.  I can get Techne or Precision on disc
ount and wonder if anyone out there has experience with these machines?
From: MPB@CBR.DWE.CSIRO.AU (Mark Bradley)
Newsgroups: bionet.molbio.methds-reagnts
Subject: protein expression systems
Date: 8 Oct 92 18:56:07 GMT
Distribution: bionet

We have been using the Maltose-Binding System over the last year
with success except in one area which I think is worth mentioning.
One can cleave the MBP from the protein of interest with factor Xa.
One is then supposed to be able to to separate the cleaved
MBP from the target protein by passing the cleaved mix over the amylose affinity
column.  The protein of interest should pass through and the MBP should be retained.
We find that after cleavage, the MBP fails to bind to the column.  I have talked
with colleagues about this and those using the same system have had identical
problems.
We have attempted to alter the affinity column parameters with no
success.  I would be interested in hearing from others who may
have experienced this with the system, and espcially those
who have been able to overcome it.

Mark Bradley
CSIRO
Div. Wildlife & Ecology
Canberra
AUSTRALIA
Newsgroups: bionet.molbio.evolution
From: mrl775@tigger.jvnc.net
Subject: Tree of Life in electronic form?
Followup-To: bionet.molbio.evolution
Reply-To: mrl775@tigger.jvnc.net
Organization: TheNews 
Date: Sun, 11 Oct 1992 14:39:19 GMT

Hello

Does anyone out there know if the "tree of life", or the complete phylogenetic
tree (based on rRNA I guess) is available in electronic form.  The standard
tree
format would be suitable, as I can draw it from that.

Is there an FTP site, or anything?

Thanks

Keith Elliston
mrl775@tigger.jvnc.net
From: ZWIEB%JASON.DECNET@relay.the.net (Christian Zwieb)
Newsgroups: bionet.molbio.evolution
Subject: Re: tree of life in electronic form
Date: 13 Oct 92 04:29:16 GMT
Distribution: bionet

Send an e-mail containing the word 'help' to rdp@mcs.anl.gov for
instructions to get a tree of life based on small subunit ribosomal rnas.
-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: kristoff@NET.BIO.NET (Dave Kristofferson)
Newsgroups: bionet.molbio.evolution
Subject: test 1
Date: 14 Oct 92 17:38:56 GMT
Distribution: bionet


test of mol-evol@net.bio.net
Newsgroups: bionet.molbio.evolution
From: mrl775@tigger.jvnc.net
Subject: Re: tree of life in electronic form
Followup-To: bionet.molbio.evolution
Reply-To: mrl775@tigger.jvnc.net
Organization: TheNews 
Date: Tue, 13 Oct 1992 20:11:30 GMT

In article <01GPVLFXA5YQ0000FM@nic.the.net> ZWIEB%JASON.DECNET@relay.the.net
(Christian Zwieb) writes:
>Send an e-mail containing the word 'help' to rdp@mcs.anl.gov for
>instructions to get a tree of life based on small subunit ribosomal rnas.
>-Chris
>

I checked this out, and sure enough, there is an FTP server there.  But, when I
downloaded the small subunit tree, it only contained the bacteria!!!  

Does anyone know where I can find a small subunit tree that contains all
species?  Do I have to go to another type of tree?  Any help on this would be
appreciated.

THanks

Keith
From: D.Hines@daresbury.ac.uk ("D.Hines")
Newsgroups: bionet.molbio.evolution
Subject: Test, please ignore
Date: 16 Oct 92 16:31:16 GMT
Reply-To: D.Hines@daresbury.ac.uk
Distribution: bionet
Original-To: mol-evol@daresbury, bioforum@daresbury

Test, please ignore
From: kristoff@net.bio.net (David Kristofferson)
Newsgroups: bionet.molbio.evolution
Subject: test 3
Date: 14 Oct 92 17:40:33 GMT
Distribution: bionet
Organization: BIOSCI International Newsgroups for Biology

test of bionet.molbio.evolution news-to-mail
From: bishop@MAP.MARC.USDA.GOV
Newsgroups: bionet.molbio.evolution
Subject: New Subscription
Date: 9 Oct 92 13:46:49 GMT
Distribution: bionet

Please enter my subscription to your list.
Thanks.
Michael D. Bishop
%USMARC-ARS
PO Box 166
Clay Center, NE 68933
(402)762-4367
Newsgroups: bionet.molbio.embldatabank
From: James Petts <pettsj@visigoth.demon.co.uk>
Subject: EMBL CD-ROM
Organization: No Affiliation
Date: Wed, 14 Oct 1992 10:03:09 GMT
X-Useragent: Nuntius v1.1

Can somebody let me know how to order and subscribe to the EMBL CD-ROM, 
please?

James Petts

********************************************************************
* "We use the classical theory on Mondays, Wednesdays and Fridays, *   
*  and the quantum theory on Tuesdays, Thursdays and Saturdays."   *
************************************** Wm. Bragg *******************
From: mlaitinen@sara.cc.utu.fi
Newsgroups: bionet.molbio.hiv
Subject: Immunity against HI virus?
Date: 11 Oct 92 16:15:24 GMT
Organization: University of Turku, Finland


I've read in Finnish Text TV that there has been found people (in the USA?)
with signs of HI virus but the test result has been negative. Some scientists
have made conclusions that those people would be able to reject the virus.

It would be interesting to hear more about this; so, are there scientists
reading this group who could tell me more about this item?

I have already 2-3 years ago insisted that there is evolution in HI virus as in
all types of virus, and it is only a question of time till scientists will find
several types of HI virus making it more difficult to find a vaccine against
it. And: last year we heard the first reports about different types of HI
virus.

I've also insisted that natural selection might influence in mankind so, that
in time there would be selected people who have an immunity against the HI
virus. What does the experts think? Could it be feasible that there, by
mistake, really exist people who have an inborn ability to reject and destroy
the HI virus?

This being a fact, mankind would be saved by means of evolution: people with an
inborn ability to reject the virus would produce more offspring. In a most
gloomy scenery the HI virus would spread little by little and contaminate all
people with no immunity and only those with an inborn immunity would produce
offspring living old enough to produce own children.

Markku Laitinen
The University of Turku
Finland
From: byrne@soma.umdnj.edu (Bruce Byrne)
Newsgroups: bionet.molbio.hiv
Subject: Recombinant DNA strategies
Summary: DNA-based strategies to combat HIV infection
Keywords: HIV,AIDS,DNA
Date: 14 Oct 92 19:52:39 GMT
Distribution: bionet

In hopes of starting a conversation....

Two groups have published results of suicide construct strategies now
frequently being applied in genetic therapeutic approaches.  See
Caruso et al. PNAS 89:182-186 (1992) and Venkatesh et al. PNAS 87:
8746-8750 (1990) for details.

Briefly, they both linked an HIV-1 LTR to HSV thymidine kinase (HSV1-TK)
and upregulated TK in first (1990) a tat expression system carried by
HeLa or Jurkat cells and then in CD4+ lymphoid cells subsequently infected
by HIV.  In either case, tat expression rendered the cells differentially
sensitive to acyclovir (or gancyclovir).  In the later paper, the in vitro
response appeared to be consistent with pharmacologically possible doses.

I've been waiting for people to use the LTR, and have wondered if others
have comments or strategies they might want to develop from the armchair...

To start:
1.  I presume that the biggest generic hurdle with this approach will be
consistent with that seen for all gene therapies reported to date: long-
term expression of the construct in vivo.  Does anyone have experience with
CD4+ target cells for reporter genes like beta-gal?

2.  What other sequences would one like to see downstream of the LTR?
Would an antisense or ribozyme sequence produce sufficient numbers of
copies to be effective?

3.  The LTR approaches all are meant to kill/control a cell containing
a good (from ITS point of view, I guess) provirus and keep it from
making more.  Anyone out there thinking about strategies to block the
life cycle with a recombinant strategy during the inward flow of the 
life cycle?


The discussion is open.  Perhaps we'll do better than the American
vice presidential candidates did on TV last night.....

Bruce Byrne
Robert Wood Johnson Medical School - Camden NJ

****usual disclaimers.....and more******
From: ballms@ctrvx1.vanderbilt.edu (BALLMS)
Newsgroups: bionet.molbio.hiv
Subject: g p 120 vaccine
Keywords: old news?
Date: 14 Oct 92 04:31:00 GMT
Organization: Vanderbilt University
News-Software: VAX/VMS VNEWS 1.41

I'm an M.T., headed for biotechnology ,
 and just noticed that here at Vanderbilt they
are asking for volunteers for the GP 120 Vaccine. Is this 
being done at other places also?  
Mark
From: ST403161@BROWNVM.BROWN.EDU (MIKE NEWSTEIN)
Newsgroups: bionet.molbio.hiv
Subject: MEETINGS
Date: 9 Oct 92 19:57:14 GMT
Distribution: bionet

HELLO

ARE THERE ANY HIV MOLECULAR BIOLOGISTS OUT THERE ANYMORE?

I WAS THINKING ABOUT GOING TO THE KEYSTONE HIV MEETING IN COLORADO THIS MARCH.
HAS ANYBODY BEEN TO THOSE MEETINGS. I WAS WONDERING HOW MUCH BASIC RESEARCH
(EG VIRAL GENE REGULATION) IS COVERED. WHAT ARE THE POSTER SESSIONS LIKE?
ARE THERE ANY OTHER MEETINGS THAT PEOPLE WANT TO DISCUSS?

MIKE NEWSTEIN
BROWN UNIVERSITY
From: rybicki@uctvax.uct.ac.za
Newsgroups: bionet.molbio.hiv
Subject: Re: Immunity against HI virus?
Date: 14 Oct 92 08:04:57 GMT
Organization: University of Cape Town

In article <1992Oct11.110901.1@sara.cc.utu.fi>, mlaitinen@sara.cc.utu.fi writes:

> inborn ability to reject the virus would produce more offspring. In a most
> gloomy scenery the HI virus would spread little by little and contaminate all
> people with no immunity and only those with an inborn immunity would produce
> offspring living old enough to produce own children.

But that is an unrealistic scenario in that risk groups at present in realistic
terms are heterosexually-active people who are promiscuous, and babies who are
unfortunate enough to get it from their mothers.  As with any other disease
which spreads by the same route (eg Hep B), there are limits to spread,
otherwise we would all already be infected with Hep B which is more infectious
and reaches much higher titre in the same body fluids than HIV. 
Epidemiologists are not nearly as gloomy about HIV as TV documentary makers and
people who have to sell newspapers.
 _________________________________________________________________________
|                                 |                                       |
|  Ed Rybicki                     |    Now you've got the hang of it      |
|  Dept Microbiology              | There's nothing you can't do with it  |
|  University of Cape Town        |         If you're very into it        |
|  PB Rondebosch 7700             |         You can't go wrong....        |
|  South Africa                   |                                       |
|  fax 27 21 650 4023             |              - Mad John               |
|  ed@micro.uct.ac.za             | (Ogden's Nut-Gone Flake, Small Faces) |
|_________________________________|_______________________________________|
Newsgroups: bionet.molbio.hiv
Fri, 16 Oct 1992 08: 55:18 +0100
               Thu, 15 Oct 1992 09: 02:41 +0100
               Thu, 15 Oct 1992 11: 03:22 +0100
From: suter@VAX.MPIZ-KOELN.MPG.dbp.de
Subject: re:hiv
Content-Identifier: 12
Original-To: hiv-molbiol@uk.ac.daresbury
Conversion: Prohibited
Distribution: bionet
Date: Thu, 15 Oct 1992 10:03:22 GMT

ed rybicki wrote:
 
+In article <1992Oct11.110901.1@sara.cc.utu.fi>, mlaitinen@sara.cc.utu.fi 
+writes:
+
+> inborn ability to reject the virus would produce more offspring. In a most
+> gloomy scenery the HI virus would spread little by little and contaminate all
+> people with no immunity and only those with an inborn immunity would produce
+> offspring living old enough to produce own children.
+
+But that is an unrealistic scenario in that risk groups at present in realistic
+terms are heterosexually-active people who are promiscuous, and babies who are
+unfortunate enough to get it from their mothers.  As with any other disease
+which spreads by the same route (eg Hep B), there are limits to spread,
+otherwise we would all already be infected with Hep B which is more infectious
+and reaches much higher titre in the same body fluids than HIV.
 
i think you should also be very carefull about trying to predict how HIV will
evolve in the future: no scientist has been able to do this. 
it is almost impossible to do so, since there are just to many
parameters that control evolution (HIV and general). so don't believe anyone who
says "HIV will evolve so and so because of this and that". show me (all !)
the data for such a statement !

+Epidemiologists are not nearly as gloomy about HIV as TV documentary makers and
+people who have to sell newspapers.

the only certainty is that many more will have to die, especially in the third 
world.....

clemens
===============================================================================
Clemens Suter-Crazzolara, PhD
Max-Planck-Institut fuer Zuechtungsforschung
Abteilung Genetische Grundlagen der Zuechtungsforschung
Carl-von-Linne Weg 10
5000 Koeln 30
Tel. xx49-221-5062.221           fax. xx49-221-5062.21
suter@vax.mpiz-koeln.mpg.dbp.de
===============================================================================
       all job offers should include a self-addressed, stamped envelope
===============================================================================
From: byrne@soma.umdnj.edu (Bruce Byrne)
Newsgroups: bionet.molbio.hiv
Subject: comments on molecular strategy (fwd)
Summary: forward of alternative HIV molecular therapies
Keywords: hiv,AIDS,DNA
Date: 16 Oct 92 18:05:37 GMT
Distribution: bionet

From POSTMASTER@dlgm.dl.ac.uk Fri Oct 16 11:22 EDT 1992
Received: from dlgm.dl.ac.uk by soma.umdnj.edu with SMTP
	(16.6/15.6) id AA17063; Fri, 16 Oct 92 11:21:56 -0400
Return-Path: <POSTMASTER@dlgm.dl.ac.uk>
Via:   UK.AC.CAMBRIDGE.BIOLOGY.MOLECULAR-BIOLOGY-1;  Fri, 16 Oct 92 16:25 WET (V41 at UK.AC.DARESBURY.DLGM)
Date:           Fri, 16 Oct 92  16:28 BST
From: CC101@UK.AC.CAMBRIDGE.BIOLOGY.MOLECULAR-BIOLOGY-1
To: BYRNE%SOMA.UMDNJ.EDU@UK.AC.DARESBURY.DLGM
Subject:        RE: Recombinant DNA strategies
Status: RO

As I cant seem to get directly onto the HIV board, I'm trying this approach
With regard to your comments on DNA strategies here are some ideas that were
part of a grant turned down by the MRC. I would think that one would need to
try at least two constructs with the LTR. Firstly, the best viral gene for an
antisense construct I would think should be the rev gene, being an important control gene
Secondly given the possible importance of apoptosis in the pathogenesis of AIDS
a LTR-bcl-2 gene construct would really be interesting (though I must say that
the speculative nature of this approach was the reason given for the grant rejection.
If you get this reply you may think of posting it onto the HIV board
From: byrne@soma.umdnj.edu (Bruce Byrne)
Newsgroups: bionet.molbio.hiv
Subject: Re: MEETINGS
Date: 12 Oct 92 17:32:35 GMT
Distribution: bionet

ST403161@BROWNVM.BROWN.EDU (MIKE NEWSTEIN) writes:


>ARE THERE ANY HIV MOLECULAR BIOLOGISTS OUT THERE ANYMORE?

	Maybe, but it's hard to tell based on this group's
	activity.   I have a theory: we're all so concerned about that
	bit of our research that may be cutting edge, we don't want
	to talk about it just yet...  Might I suggest that anyone
	having some ideas that ther're not going to be prusuing themselves
	might share them.  I'll try a separate post tomorrow and see if
	it flies...

>I WAS THINKING ABOUT GOING TO THE KEYSTONE HIV MEETING IN COLORADO THIS MARCH.
>HAS ANYBODY BEEN TO THOSE MEETINGS. I WAS WONDERING HOW MUCH BASIC RESEARCH
>(EG VIRAL GENE REGULATION) IS COVERED. WHAT ARE THE POSTER SESSIONS LIKE?
	
	Great meetings, even if you don't ski.  Good mix of experts and
	real folks.  Meetings much more oriented toward basic research
	than, say, your average international megaconference.  I spent
	a long time at my first poster there explaining techniques to a
	grad student while an older fellow waited patiently to ask
	his question.  Turned to talk to him.  Jonas Salk; great questions.
	Oops.

	By the way, take time to ski, or learn


Bruce Byrne
RWJMS-Camden
Newsgroups: bionet.molbio.hiv
From: wij@world.std.com (William I Johnston)
Subject: Re: Just a Question
Organization: The World Public Access UNIX, Brookline, MA
Date: Thu, 8 Oct 1992 03:45:57 GMT