HDTenEightyP/NetNews huggingface dataset:
From: fwu@CABELL.VCU.EDU (Fang-Sheng Wu)
Newsgroups: bionet.molbio.gdb
Subject: (none)
Date: 5 Nov 92 20:42:08 GMT
Distribution: bionet

MOLECULAR GENETICIST


The Department of Biology at Virginia Commonwealth University
invites applications for a tenure-track Assistant Professor
position.  Teaching responsibilities will include undergraduate
genetics and, periodically, upper/lower level courses in the
candidate's area of specialty. Candidates must hold a Ph.D. in
genetics or a closely related discipline; post-doctoral
experience is preferred.  The successful candidate will be
expected to develop an externally-funded research program in an
area of molecular genetics complementary to existing
environmental and plant genetics research programs.  Virginia
Commonwealth University is a culturally diverse institution with
an enrollment of 22,000 students.  The Biology Department
includes 26 faculty members and has a M.S. graduate
program.  Submit curriculum vitae, selected reprints, statements
of research and teaching interests, and three letters of
recommendation by 21 December 1992 to:  Dr. John F. Pagels, Dept.
of Biology, Virginia Commonwealth University, Richmond, VA  
23284-2012.  Virginia Commonwealth University is an Equal
Employment Opportunity/Affirmative Action Employer and encourages
women, minorities and persons with disabilities to apply.
From: bsh@MED.PITT.EDU (Basavaraju Shankarappa)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re:  immunopptn of RNA/ribosome complexes
Date: 12 Nov 92 23:49:17 GMT
Distribution: bionet

Regarding the immunoprecipitation of RNA/ribosome complex:
	Look for the article "use of antibodies to obtain specific polysomes"
by Dennis Lynch in Methods Enzymol : Guide to Molecular Cloning Techniques
BC  vol 152: 248-253.  I think he covers most the topics you were interested
I have not tried it.  Good luck if you do.
Raj Shankarappa
bsh@med.pitt.edu
From: rosswhet@forbt2.nrrc.ncsu.edu (Ross Whetten)
Newsgroups: bionet.molbio.methds-reagnts
Subject: G/C tails in expression constructs
Date: 11 Nov 92 18:08:00 GMT
Organization: North Carolina State University

Hello netters!
I am making an expression construct with a "constituitive" promoter driving
transcription of a full-length cDNA that was originally cloned using G/C 
tailing at a Pst I site. Are the G/C tails likely to present problems for
the expression of the fusion gene? There are likely to be 20 to 50 consecutive
G residues in the 5' untranslated portion of the resulting mRNA. This is
a plant construct using the CaMV 35S promoter, if that makes a difference.
Comments from anyone with experience in this issue are welcome, as are any
references to papers that address the question. Thanks.
Ross
rosswhet@unity.ncsu.edu
From: agoodrid@vaxa.weeg.uiowa.edu (Stephen Klautky)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: sequencing from 300-500 bp
Date: 13 Nov 92 03:34:00 GMT
Organization: University of Iowa, Iowa City, IA, USA
News-Software: VAX/VMS VNEWS 1.41

In article <1992Nov12.150858.16911@magnus.acs.ohio-state.edu>, rrumpf@magnus.acs.ohio-state.edu (Robert Rumpf) writes...
>There is a product called "Long Ranger" which substitutes for the acrylamide 
>gels.  It is supposed to do exactly what you asked for, give resolution on the 
>order of 500-800 (?? been a while since I've heard about it) bases per gel.  
>Several people in our department have tried it; only one has gotten it to work.
>Everyone else had technical problems either pouring such a gel or taking it off
>the glass.
> 
>Bob

Some of the people in our lab use/have tried the Long Ranger
gel mix.  Sometimes it does seem to be harder to get the gel
off the plate onto the gel backing paper than the usual
A/B gel.  Generalization - It seems that if you have a little
trouble with sticky gels on plates with the regular mixes then
your troubles will increase; if you have no such problems then
Long Ranger should not give you problems.  This is just my
impression your observations may be different.

One annoying problem which has occured a few times - the gel
does not stick to the gel backing paper!  I had to put the
paper on the gel, invert, carefully lift the glass plate off
leaving the gel on the paper.

From what I can see the Long Ranger has two major advantages:
First, it runs faster than the standard mixes.  Unfortunately,
we did not see the advertised increase in resolution - but you
know how that can vary - almost day to day.  Secondly, you don't
fix the gel.  Of course, I know it has become fashionable not
fix the ordinary gels (but in my experience this practice gives
you a lower signal).

Some of my collegues had major problems with the LR gel sticking
to the long plate and complain a bit.  I have good plates and the
whole process goes easy for me (mostly) - except when people
walk off with my plates.  :-)

Steve     AGOODRID@VAXA.WEEG.UIOWA.EDU
From: mic296e@vaxc.cc.monash.edu.au
Newsgroups: bionet.molbio.methds-reagnts
Subject: re:Aqua Bidest
Date: 6 Nov 92 03:52:26 GMT
Organization: Computer Centre, Monash University, Australia

Aqua bidest may just mean "double destilled water". Some funny german
speaking people (as myself) use this expression I think.
From: ARF6%manvax@CUVMB.CC.COLUMBIA.EDU ("FRANCES M. CARDILLO")
Newsgroups: bionet.molbio.methds-reagnts
Subject: subscription
Date: 11 Nov 92 01:38:00 GMT
Distribution: bionet

unsubscribe
Newsgroups: bionet.molbio.methds-reagnts
From: C-Y Khoo <cyk10@cus.cam.ac.uk>
Subject: anti-GST antibodies for fusion protein detection
X-Xxmessage-Id: <A7294D2466022D4A@cy-mac.welc.cam.ac.uk>
X-Xxdate: Fri, 13 Nov 92 12:16:36 GMT
Organization: Wellcome/CRC Institute, Cambridge
X-Useragent: Nuntius v1.1.1d11
Date: Fri, 13 Nov 1992 12:16:36 GMT

I too, have been looking for anti-GST antibodies, but without any success.
I called Pharmacia and they said that they are in the process of
developing
such a reagent, but that it won't be ready for some time yet. There are
commercial sources of anti-GST antibodies, but they're raised against the
human antigen, and according to Pharmacia, don't cross react with the S.
japonicum antigen. Your best bet is to express pGEX, purify GST from
the supernatant, and immunize a rabbit with it.

Depending on various things, I _may_ have some anti-GST by January next
year
(I'm currently immunizing with a fusion protein). Ask me later!

Regarding your expression problems, I found out that _most_ fusion
proteins
are insoluble and are found in inclusion bodies, _not_ the supernatant (as
stated in Smith and Johnson (or indeed, Current Protocols in Molecular
Biology). See Fiona Marston's articles in Biochem J. (1986) 240, 1-12 and
an article of the same name in another journal (don't have the reference
at hand, unfortunately!). Also, Nagai and Thogersen (1987) Methods Enzymol
153, 461-481 for inclusion body prep. As a first step, you may want to
boil some of the pellet to see if the fusion protein is actually being
produced, but in an insoluble form.

Good luck!

=======================================================================
=======
Chong-Yee Khoo                             E-mail: cyk10@cus.cam.ac.uk
Wellcome/CRC Institute                     Talk:  
cyk10@cy-mac.welc.cam.ac.uk
Tennis Court Road                          Phone:  +44 223 334110
Cambridge                                  Fax:    +44 223 334089
CB2 1QR
From: dubear@molbio.cbs.umn.edu (Dubear Kroening)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Yeast X-gal minimal media plates?
Date: 12 Nov 92 23:16:14 GMT
Organization: University of Minnesota
X-Newsreader: Tin 1.1 PL5


Hi netters,

I hope someone can help me.  We are trying to make selective plates (SD) for
yeast that contain X-gal and a nonfermentable carbon source (glycerol, ethanol,
galactose, etc...).  We can get good growth and color formation using selective
plates containing glucose, but when we try a nonfermentable carbon source, we
get no growth.  We have searched the literature extensively, but can find no
references for what we want.  Does this mean it's not possible?  Any info, ideasor other suggestions would be welcomed greatly.  If you don't want to post you
can email me directly   dubear@molbio.cbs.umn.edu

Thanks in advance,

Dubear
From: karsten@biogene1.uni-bielefeld.de (Karsten Niehaus)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re:Aqua Bidest
Date: 5 Nov 92 11:50:29 GMT
Distribution: bionet
Mailer: Elm [revision: 64.9]
Original-To: methods@uk.ac.daresbury


> 
> A researcher here has an HPLC procedure from a German paper that calls for
> "Aqua Bidest" as part of the solvent system.  We've so far been unable to
> determine what this is -- can anyone help us?
> 
> Thanks
> ________________________________________________________________________________
> The opinions stated are mine, not those of NCTR or its sponsoring organizations.
> 
> Bill Melchior                                ||     OMNISCIENCE
> National Center for Toxicological Research   ||    Knowing what
> Jefferson, AR  72079                         ||    thou knowest not
> (501) 543-7206                               ||    is in a sense
>                                              ||    omniscience.
> WMELCHIOR@NTET.NCTR.FDA.GOV                  ||       from Grooks, Piet Hein
> 
> 
> 
Dear Bill Melchior,

I think you got the recipe for "Aqua Bidest" (correct: Aqua bi destillata).
Since some years I avoid "Aqua Bidest" because it contains more organic 
traces (due to the destillation). If you use a UV-Monitor at 180 to 220 nm
in your HPLC system this may cause problems. We found that "Aqua Demin"
(deminralized water) works better. 

Karsten Niehaus
University odf Bielefeld
Biology-Genetic
4800 Bielefeld
Germany

e-mail: karsten@biogene1.uni-bielefeld.de
From: moseley@u.washington.edu (Steve L. Moseley)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: In vitro mutagenesis kit recommendation?
Date: 13 Nov 92 21:53:48 GMT
Organization: Microbial Pathogenesis and Motorcycle Maintenance

In article <BxMKJB.Lz8@usenet.ucs.indiana.edu> jgraham@bronze.ucs.indiana.edu (the End) writes:
>From: jgraham@bronze.ucs.indiana.edu (the End)
>Subject: Re: In vitro mutagenesis kit recommendation?
>Date: Thu, 12 Nov 1992 22:45:10 GMT

>In <1dr98rINNcdb@usenet.INS.CWRU.Edu> ca566@cleveland.Freenet.Edu (Michael Holloway) writes:


>>I apoligize if I'm lowering the intellectual level of this conference :-)
>>but I promised to post an inquiry to see if there are any favorite kits for
>>mutagenesis.  Is there any difference in the products?  Is there a
>>consensus on a favorite?

>>Thanks
>>Mike

>You are lowering the value of this forum. About half of the traffic here 
>is based on discussions of what is the best kit to "buy" rather than what
>is really important, the technical details of the procedures. 

>As with Exo III deletions, get yourself a copy of Maniatis, a few papers
>from the library (I recommend the Kunkel procedure) and yes, buy a few 
>salts from the stockroom. You don't need the high priced hand-holding
>from Biosham (TM). 

>Then come back with some intelligent questions, and I'm sure you will find
>more than enough help here.

>Cheers,

>Jim
>J. Graham
>Biology and Chemistry Departments
>Indiana University -Bloomington

Sorry Jim, but posts like yours aren't cause for any cheers. Your answer 
helps no one. Find another alt. or rec. group to vent your spleen and leave 
this group for those interested in learning and helping.

As to the original question, we've used the Kunkel procedure with salts we 
bought from the stockroom with some success. However, on the recommendation 
of a colleague whose intellectual level is lower than Dr. Graham's, we tried 
the T7-GEN in vitro mutagenesis kit from US Biochemical. We've been 
extremely pleased with its efficiency. A reference is Vandeyar et al., Gene 
65:129, of course those of us who use the K word probably don't know the way 
to the library. Mutant selection is based on methylation of the mutant 
strand, nicking of the original strand by restriction enzyme, and removal of 
the nicked original strand by ExoIII. It has worked very nicely for us. Most 
plaques are mutants with the kit, while in our hands, the Kunkel procedure 
efficiency was around 10%. 

 - Steve

 - Steve
__________________________________________________________________________
Steve L. Moseley                                  moseley@u.washington.edu
Microbiology SC-42                                Phone: (206) 543-2820
University of Washington                          FAX:   (206) 543-8297
Seattle, WA 98195
From: cytogen@chmeds.ac.nz (Martin Kennedy)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Northern Blots
Date: 10 Nov 92 16:00:40 +1200

In article <1dknqeINNt6r@matt.ksu.ksu.edu>, jaishre@matt.ksu.ksu.edu (Jaishree Muppala Chittoor) writes:
> Help!  I am seeing a very intense background in all my blots, recently.  The 

....stuff deleted...
> 
> I changed blot (to gene screen plus), changed probe and even used a different 
> pre hyb solution (5x SSC, 5x denhart, 0.5%SDS, 20mM phosphate buffer, 300u/ml 
> salmon sperm DNA, 5mM EDTA), but nothing seems to eliminate the very intense 
> background.  I have even tried washing with water.
> 

I find GeneScreen Plus requires higher concentrations of salmon sperm DNA for
blocking than several other membranes.  If the black background doesn't wash
off then it's covalently bound to your membrane, and the membrane probably
wasn't sufficiently blocked in the first place.  Try using ca 500ug/ml ssDNA in
your prehyb mix; I also add a further 1-2mg of ssDNA to my probe when I boil
it, and then add about 10ml of hyb mix before tipping it into the bag/bottle.
As for the black membranes, put them away for a few months!

> Jaishree [jaishre@matt.ksu.ksu.edu or jaishre@ksuvm.ksu.edu]
> 
Good luck!

Martin
Newsgroups: bionet.molbio.methds-reagnts
From: rrumpf@magnus.acs.ohio-state.edu (Robert Rumpf)
Subject: Re: sequencing from 300-500 bp
Organization: The Ohio State University
Date: Thu, 12 Nov 1992 15:08:58 GMT

There is a product called "Long Ranger" which substitutes for the acrylamide 
gels.  It is supposed to do exactly what you asked for, give resolution on the 
order of 500-800 (?? been a while since I've heard about it) bases per gel.  
Several people in our department have tried it; only one has gotten it to work.
Everyone else had technical problems either pouring such a gel or taking it off
the glass.

Bob
From: preissj@CLVAX1.CL.MSU.EDU ("J Preiss--Seq Anal")
Newsgroups: bionet.molbio.methds-reagnts
Subject: re Nested deletion kits/methods
Date: 5 Nov 92 19:59:00 GMT
Distribution: bionet

Hi netters

	In response to Marc Van Ranst's request for info on nested deletion
kits / methods, I highly recoment the method in the Promega kit.
	This kit uses ExoIII and goes from the initial restriction digest
to the final transformation of E. coli with no phenol extractions and no
precipitations.  It is, therefore, very fast, easy, and convenient (not to
mention cost effective).  I have used this method with much success, and
many of my friends and colleages have used it with success as well.  I must 
add, however, that I have never actually used the kit!  Someone passed the
instruction manual around the department and we all made up the buffers 
ourselves.  Some people used Promega enzymes, but most of us used BMB
enzymes for the work.  either will do the job.
	Good luck with your deletions.

Dr. Leonard N. Bloksberg
PreissJ@clvax1.cl.msu.edu
Dept. of Biochemistry
Michigan State University
From: axa12@po.cwru.edu (Ashok Aiyar)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: MEDLINE
Date: Sun, 8 Nov 1992 00:06:44 GMT
Organization: CWRU School of Medicine
Distribution: bionet
NNTP-Posting-Host: axa12-slip.dialin.cwru.edu
Summary: Grateful Med works over TCP/IP and SLIP connections
Keywords: Grateful Med, Internet, TCPIP

In article <axa12.41.721174772@po.cwru.edu> axa12@po.cwru.edu (Ashok Aiyar) writes:

>>This may be common knowledge, or it may have been asked before, but I don't
>>know how to access, if possible, MEDLINE via gopher or telnet (preferably 
>>for free). I would if somebody out there knows how to do this please let 
>>me know 
>
>MEDLINE can be accessed over the Internet (via Telnet) but I do not believe 
>that access is free (at least we still pay for it).  For those who are 
>interested, the NLM MedLine "search" software works fine over SLIP 
>connections.  I just got it working about 10 minutes back.  The trick is to 
>use the ETHERSLIP packet driver and not the SLIP8250 driver (on DOS 
>machines).

To clarify myself -- by Medline "search" software, I am specifically 
referring to Grateful Med 6.0

Ashok
----------------------------------------------------------------------
                             Ashok Aiyar
                     Department of Biochemistry
                         CWRU Medical School
                          axa12@po.cwru.edu
----------------------------------------------------------------------
From: eric@bcserv.wustl.edu (Hugo (Eric))
Newsgroups: bionet.molbio.methds-reagnts
Subject: Protein phosphorylation assays using dot/slot blot ?
Summary: Does anyone have a good protocol for 32p assays?
Keywords: 32p, protein, slot blots
Date: 13 Nov 92 22:38:31 GMT
Organization: Washington University in Saint Louis, MO USA
NNTP-Posting-Host: bcserv.wustl.edu

Dear Bionetters,
     Can anyone recommend a protein phosphorylation assay protocol
that uses a slot/dot blot manifold?  I've been using the std. TCA ppt
method onto glass filters or Whatman paper and it is a bit tedious
with 80 - 90 samples per experiment.  Basically, I need to know if anyone
has had sucess with PVDF or some other membrane.  I would like to
use a beta counter/scanner to quantitate the results.  Any info would
be appreciated.  Please email responses, I will post a summary if requested.
Thanks.                            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
From: MPB@CBR.DWE.CSIRO.AU (Mark Bradley)
Newsgroups: bionet.molbio.methds-reagnts
Subject: manuals for MaClade & Treedraw
Date: 11 Nov 92 11:48:06 GMT
Distribution: bionet

Can anyone point me to where I can FTP the user manuals for
the programs MacClade and TreeDraw? 

thanks in advance

Mark Bradley
CSIRO, Div. wildlife & Ecology
Canberra
Australia
EMAIL: mpb@cbr.dwe.csiro.au
From: vrati@dbe.csiro.au (Sudhanshu Vrati)
Newsgroups: bionet.molbio.methds-reagnts
Subject: site-directed-mutagenesis
Date: 11 Nov 92 22:46:05 GMT
Distribution: bionet


I have been doing site directed mutagenesis of double stranded plasmids using the unique restriction site elimination procedure very successfully until recently
when I ran into troubles- getting no mutants at all.  I have previously used
BMH71-18mutS and GM119 (dcm and dam minus) cells successfully but now nothing
seems to work.  Any one out there with helpful suggestions ?  Actually I want
to mutagenise a 16KB plasmid.  Is there any other good method of mutating
such big plasmids ?
Thanks a lot for the help.
Sudhanshu Vrati
CSIRO Div. Biomolecular Engg.,
Sydney (Australia)
From: suter@VAX.MPIZ-KOELN.MPG.dbp.de
Newsgroups: bionet.molbio.methds-reagnts
Subject: aqua bidest
Date: 5 Nov 92 10:04:17 GMT
Distribution: bionet
Content-Identifier: 12
Original-To: methods@uk.ac.daresbury
Conversion: Prohibited



aqua = H2O
bi = 2 or twice
dest = distilled 

aqua bidest is twice distilled water, no to be confused with VE water
which is only once destilled.


clemens
From: bsh@MED.PITT.EDU (Basavaraju Shankarappa)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re:  Which salt of X-P to use for detection of alkaline phosphatase?
Date: 12 Nov 92 16:02:59 GMT
Distribution: bionet

We used to use the following as substrates for Western blotting.
Yields a bright pinkish band which quite pleasing to the eye and slide-
making but may be not that high contrast for the photography.

100 mg Fast Red TR salt (Sigma: f8764)
50 mg Napthol AS-MX phosphate (Sigma N5000)
              ^^^^^^^^^^^^^^^
Each of these are dissolved in 50 ml of 0.5M tris pH 8.0, mixed in the 
tray and incubated with the membrane. 

Worked well for me.
Raj Shankarappa
bsh@med.pitt.edu
From: wmelchior@ntet.nctr.fda.gov
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Gel Slick anyone?
Date: 13 Nov 92 08:45:31 -0600
Organization: NATIONAL CENTER FOR TOXICOLOGICAL RESEARCH

> My wife and I (work in the same lab :-) ) were curious if anyone has
> tried the AT Biochem Gel Slick as an alternative to Pierce's Surfacil.
> Pierce's product works well but the HCl fumes generated can be irratating.

If you're just using it to treat electrophoresis plates, as opposed to 
things like vessels and pipet tips for use in reactions, try Rain-X.  It's 
sold for making auto windshields water-repellant.  As near as I can tell, 
it's just a solution of a hard wax in a volatile solvent.  It's 
non-irritating and probably not very toxic.  It's available cheaply in the 
auto departments of discount stores.
________________________________________________________________________________
The opinions stated are mine, not those of NCTR or its sponsoring organizations.

Bill Melchior                                ||     OMNISCIENCE
National Center for Toxicological Research   ||    Knowing what
Jefferson, AR  72079                         ||    thou knowest not
(501) 543-7206                               ||    is in a sense
                                             ||    omniscience.
WMELCHIOR@NTET.NCTR.FDA.GOV                  ||       from Grooks, Piet Hein
From: jgraham@bronze.ucs.indiana.edu (the End)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: In vitro mutagenesis kit recommendation?
Date: 12 Nov 92 22:45:10 GMT
Organization: Indiana University

In <1dr98rINNcdb@usenet.INS.CWRU.Edu> ca566@cleveland.Freenet.Edu (Michael Holloway) writes:


>I apoligize if I'm lowering the intellectual level of this conference :-)
>but I promised to post an inquiry to see if there are any favorite kits for
>mutagenesis.  Is there any difference in the products?  Is there a
>consensus on a favorite?

>Thanks
>Mike

You are lowering the value of this forum. About half of the traffic here 
is based on discussions of what is the best kit to "buy" rather than what
is really important, the technical details of the procedures. 

As with Exo III deletions, get yourself a copy of Maniatis, a few papers
from the library (I recommend the Kunkel procedure) and yes, buy a few 
salts from the stockroom. You don't need the high priced hand-holding
from Biosham (TM). 

Then come back with some intelligent questions, and I'm sure you will find
more than enough help here.

Cheers,

Jim
J. Graham
Biology and Chemistry Departments
Indiana University -Bloomington
From: vrati@dbe.csiro.au (Sudhanshu Vrati)
Newsgroups: bionet.molbio.methds-reagnts
Subject: (none)
Date: 11 Nov 92 22:38:20 GMT
Distribution: bionet

I had been doing site dirested mutagenesis of double stranded plasmids using
the unique restriction site elimination procedure very successfully until
recently when I ran into troubles- getting no mutants at all.  I have previously
used BMH71-18mutS and GM119 (dcm and dam minus) cells successfully but now 
nothing seems to work.  Any one out there with helpful suggestions ?  Actually
I want to mutagenise a 16KB plasmid.  Is there any other good method of 
mutagenising such a big plasmid ?
Thanks a lot for the help.
Sudhanshu Vrati
CSIRO Division of Biomolecular Engg.,
Sydney (Australia)