HDTenEightyP/NetNews huggingface dataset:
From: pnj@stein.u.washington.edu (JnP)
Subject: Help: Panning
Organization: University of Washington, Seattle
Date: Sat, 12 Dec 1992 01:23:30 GMT

Hi there, I would like to know how many surface proteins have people 
successfully used for cell panning.  Could any expert on this provide 
me with some information (also for panning in general)?  I will post 
replies I get.  

Many thanks!

-Fred
From: nguyenhd@HERMES.BC.EDU ("NGUYENHD")
Newsgroups: bionet.molbio.methds-reagnts
Subject: IgM for affinity chromatography
Date: 11 Dec 92 20:14:22 GMT
Distribution: bionet

I have purified IgM antibody from my culture media by Sephacryl 300.  I have
also tried to couple this antibody to CNBr-activated Sepharose 4B.  Antibody
binds to the beads, but it loses its activiy and leaks with the eluting buffer
(0.1M Glycine, pH 2.5).  How do I get around these problems?

Thank you in advance.

Hung Nguyen
Biology Dept., Boston College
NGUYENHD@hermes.bc.edu
From: danj@WELCHGATE.WELCH.JHU.EDU (Dan Jacobson)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: protein seq. alignment programe
Date: 18 Dec 92 22:14:45 GMT
Distribution: bionet



In article <9212180511.AA27010@pelican.dbe.csiro.au> vrati@dbe.csiro.au (Sudhanshu Vrati) writes:
>I am trying to align more than three protein sequences using gcg pile up
>programe.  Are there any other programes available for this purpose ?
>Thanks for your response,
>Sudhanshu
>

Yes there are several programs that help you do multiple alignments here 
are a three of them - ClustalV, Treealign, and Macaw.


Clustal(V) is a popular aligner that is available for Unix, VMS, DOS, and
Macs.  You can get it by anonymous ftp from several places I'll list one here:

Host evolution.bchs.uh.edu

    Location: /pub/gene-server/dos
           FILE -rwxr--r--      91153  Sep 16 1991  clustalv.uaa
           FILE -rwxr--r--      91156  Sep 16 1991  clustalv.uab
           FILE -rwxr--r--      91156  Sep 16 1991  clustalv.uac
           FILE -rwxr--r--       6241  Sep 16 1991  clustalv.uad
    Location: /pub/gene-server/incoming
           FILE -rw-r--r--     199453  Sep 13 03:54  clustalv.exe
    Location: /pub/gene-server/mac
           FILE -rwxr--r--      97223  Sep 16 1991  clustalv1.hqx
           FILE -rwxr--r--      66491  Sep 16 1991  clustalv2.hqx
    Location: /pub/gene-server/unix
           FILE -rwxr--r--      91155  Sep 16 1991  clustalv.uaa
           FILE -rwxr--r--      30012  Sep 16 1991  clustalv.uab
    Location: /pub/gene-server/vms
           FILE -rwxr--r--      91154  Sep 16 1991  clustalv.uaa
           FILE -rwxr--r--      44054  Sep 16 1991  clustalv.uab


Treealign is another alignment package which is available for Unix
and VMS.  It too is available by anonymous ftp from several places - one is:

Host fly.bio.indiana.edu

    Location: /molbio/align
           FILE -rw-r--r--     298974  Jun 17 1991  treealign.shar
 

Macaw is an alignment package (can do up to 16 sequences at a time) for the 
PC with Windows 3 (or higher), you can obtain it by anonymous ftp from:

Host ncbi.nlm.nih.gov

    Location: /pub
      DIRECTORY drwxr-xr-x        512  Sep  1 20:41  macaw
    Location: /pub/macaw
    Location: /pub/macaw/util/.source
         FILE -rw-r--r--  1 1028       269238 Jul 14 21:38 macaw105.zoo
         FILE -rw-r--r--  1 1028       133876 Dec 18 16:56 macaw106.arc
         FILE -rw-r--r--  1 1028       104406 Dec 18 16:56 macaw106.lzh
         FILE -rw-r--r--  1 1028       104536 Dec 18 16:56 macaw106.zip
         FILE -rw-r--r--  1 1028       134642 Dec 18 16:56 macaw106.zoo

If you get Macaw you'll probably want to get a (hardcopy) manual which
Greg Schuler will send you if you drop him a note (schuler@ncbi.nlm.nih.gov).


Kudos go to the authors and the ftp site providers.    


Best of luck,

Dan Jacobson

danj@welchgate.welch.jhu.edu
From: bhjelle@carina.unm.edu
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: colony screening with Amersham ECL (RPN 3000 and 3004)
Date: 18 Dec 92 14:53:55 GMT
Organization: University of New Mexico, Albuquerque

In article <1992Dec16.151837@genethon.fr> bouchier@genethon.fr (Christiane Bouchier) writes:
>
>A week ago I asked whether anyone had ever carried out tests for screening
>colonies  with Amersham ECL direct system. So far I received no suggestions
>that could help me overcome the problem we have of detecting peroxydase 
>specific hybridization because of a high background.
>Once again thanks for your help !
>
For months we used Amersham ECL for a dot blot assay that is
used a lot in my lab. Backgrounds were highly variable but
eventually got so out of hand we had to switch back to
32P. We were unable to fix the problem with the blocking
procedures recommended by Amersham or any other obvious
manipulation. The problem was nonspecific blobs of signal
that covered parts of the membrane that had never seen
any biological material.

Amersham will have suggestions that you might want to
try. Contact their rep. However, we found it easier to
switch back to isotopes than to do Amersham's R&D.

Brian
From: bhjelle@carina.unm.edu ()
Subject: Re: Taq - not so promiscuous???
Date: Wed, 16 Dec 92 20:20:26 GMT
Organization: University of New Mexico, Albuquerque

In article <1992Dec15.210313.24687@news2.cis.umn.edu> horton@molbio.cbs.umn.edu (Robert Horton) writes:
>	Your observation of a low rate of incorporation of errors by Taq
>is not unusual. We found no errors in over 6000 bases from clones amplified
>from cDNAs (J. Immunol. 145:1782-7, 1990). The reaction conditions were
>"standard" except that the [Mg++] was a little low (1mM). We had slightly
>higher error frequencies in other experiments, which happened to use slightly
>more Mg. (dNTPs were always 200uM each).
>	I think Taq often gets a bum rap as far as errors introduced into
>clones is concerned. The key may be that error _frequency_ (i.e., the
>observed fraction of errors incorporated into the product) is not directly
>correlated with error _rate_ (the fraction of wrong bases incorporated
>by Taq). This is because of "thermal proofreading" (as I believe they call
>it in California), in which DNA strands that get the wrong base added don't
>get extended efficiently by polymerase, so these errors don't show up in
>the final product.
>
What gets  *measured*  in most assays is the actual rate of misincorp-
orations into the final product, and this is what most of us
care about. Some may recall that I posted last month about
a truly spectacular misincorporation rate (11 substitutions and
3 frameshifting insertions and deletions out of 511 bp sequenced).
This was with many cycles (45) using 50ng plasmid template
and 4mM MgCl2/500mM dNTP each. Apparently high dNTP, lots
of cycles, and high MgCl2 are prone to increase error rate.
That said, on a number of previous occasions I have used 45
cycles from various templates and experience negligible
error rates.

I repeated the PCR on the plasmid that was originally used
as PCR template in the experiment with high mutation rate
using much lower Mg (1.6mM) and much lower dNTP (200 each,
I think). I ran product on a gel at 10, 15 and 20 cycles;
it was increasing only about 2x with each 5 cycles -
not a very robust PCR. However, when I pooled the 10 & 15
cycle product and cloned that, the product (so far) has
been free of misincorporations etc.

So the bottom line. If you want to decrease PCR misincorporations,
(1) lower dNTP; (2) lower Mg; and (3) lower cycle #.

Brian
From: Klaus.Matthaei@anu.edu.au
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Cleaning electroporation cuvettes
Date: 16 Dec 92 23:37:40 GMT
Distribution: bionet

>Anyone have a good method for cleaning cuvettes for electroporation?
>Especially useful would be methods that remove any trace bits of DNA, yet
>do not take a toll on the lifetime of the cuvette (no flames please, I know
>they are sold for single use, only :^).  
>
>David Nunn, Ph.D.
>Department of Microbiology
>University of Illinois
>Urbana, IL 61801
>(217) 333-6131


G'Day

Rinse after use, wash with detergent, rinse, soak in 10% sodium
hypochlorite (standard bleach) 5 minutes, rinse well, dry and reuse.  This
will destroy all DNA contamination.

Cheers Klaus.
--------------------------------------------------------------------------
Klaus Matthaei
Gene Targeting
The John Curtin School of Medical Research
The Australian National University
E-mail: Klaus.Matthaei@anu.edu.au

"If you don't do: you rust"
--------------------------------------------------------------------------
From: vkapur@bcm.tmc.edu (Vivek Kapur)
Newsgroups: bionet.molbio.methds-reagnts,bionet.immunology
Subject: Need source for polyclonal anti-human collagen IV
Followup-To: poster
Date: 15 Dec 1992 16:38:18 GMT
Organization: Baylor College of Medicine, Houston, Tx
Distribution: usa
NNTP-Posting-Host: bcm.tmc.edu
Keywords: antibodies, collagen, immunology

	I am looking for a source for polyclonal anti-human collagen IV
(preferably rabbit) in order to run a couple of western immunoblots.  All
the commercial sources that I have tried only carry monoclones - which are
not suited for what I am trying to do.  Will appreciate any assistance in
this regard.  Please reply by e-mail or post to this newsgroup.

	-vivek

e-mail:  vkapur@bcm.tmc.edu or
	 vkapur@darwin.bcm.tmc.edu
From: bsh@MED.PITT.EDU (Basavaraju Shankarappa)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Immuno-affinity Chromatography
Date: 11 Dec 92 20:40:53 GMT
Distribution: bionet

> I have purified IgM antibody from my culture media by Sephacryl 300.  I have
> also tried to couple this antibody to CNBr-activated Sepharose 4B.  Antibody
> binds to the beads, but it loses its activiy and leaks with the eluting buffer
> (0.1M Glycine, pH 2.5).  How do I get around these problems?
> Hung Nguyen
> Biology Dept., Boston College
> NGUYENHD@hermes.bc.edu

	I had used affi-gel matrices for immunoaffinity purification of
antigens a while back.  About two years back BioRad started marketing
affigel HZ which is supposed to bind to the antibody at its Fc region
through carbohydrate moeities (?).  Is it worthwhile to try it?
Has anybody tried it.  The company's graphs show a considerably increased
yield.  May be it is better to trust results from some lab.  
I do not see any reason why IgM should not bind in the same way.
Raj Shankarappa
bsh@med.pitt.edu
Univ of Pittsburgh

Xref: sparky bionet.molbio.methds-reagnts:2899 bionet.general:1983
From: walden@mcclb0.med.nyu.edu (Paul Walden)
Newsgroups: bionet.molbio.methds-reagnts,bionet.general
Subject: Re: ENZO
Date: 15 Dec 92 14:52:45 GMT
Organization: NYU Medical Center, New York, NY 10016, USA

In article <1992Dec14.100535.1@vax.path.ox.ac.uk>, rpgrant@vax.path.ox.ac.uk writes:
> On behalf of a colleague...
> Has anyone heard of the company  Enzo ???
> They sell biotin 11 dUTP.
> I'm sorry, we don't have any other information.
> Thanks,
> Richard
> RPGRANT@vax.path.ox.ac.uk

Yup,

There address and phone # is

Enzo Biochem. Inc.
325 Hudson Street, 
New York
NY10013

Tel. (212) 741-3838

I used some anti-biotin antibodies from them.

Hope this helps.

Paul
*****************************************************************************
Paul Walden, Ph.D.
Department of Biochemistry
550 First Avenue
New York University Medical Center
New York 
NY 10016                                  Internet: WALDEN@MCCLB0.MED.NYU.EDU
*****************************************************************************
From: KNECHT@UCONNVM.UCONN.EDU (Dave KNecht)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Dig Southern Blocking
Date: 14 Dec 92 20:33:12 GMT
Distribution: bionet

To all you Dig chemiluminescent users-  what are you all using for blocking you
r filters prior to Ab addition.  Can you use store bought nonfat dry milk inste
ad of Boehringer's exorbitant "blocking reagent".  I am afraid of phosphatase a
ctivity in the milk and find that    NFDM cannot be autoclaved (it becomes a cl
otted mess) to kill any activity.  Anyone tried 1 or 2% NFDM without autoclavin
g?
Newsgroups: bionet.molbio.methds-reagnts
From: bouchier@genethon.fr (Christiane Bouchier)
Subject: colony screening with Amersham ECL (RPN 3000 and 3004)
Originator: biagioli@halogene.genethon.fr
Organization: Genethon -- CEPHB Human Polymorphism Study Center, Paris, France
Date: Wed, 16 Dec 1992 14:18:37 GMT


A week ago I asked whether anyone had ever carried out tests for screening
colonies  with Amersham ECL direct system. So far I received no suggestions
that could help me overcome the problem we have of detecting peroxydase 
specific hybridization because of a high background.
Once again thanks for your help !

Christiane Bouchier
Genethon
1, rue de l'Internationale
91 000 EVRY
FRANCE
Tel : 19.33.1.69.47.29.66
email : bouchier@genethon.fr
From: pakapke@IASTATE.EDU
Newsgroups: bionet.molbio.methds-reagnts
Subject: (none)
Date: 12 Dec 92 00:19:53 GMT

I want to know if any papers have been published describing 
electroporation techniques as a means of introducing phage into
bacteria instead of in vitro packaging and infecting the bacteria.
   Thanks ahead of time.
Newsgroups: bionet.molbio.methds-reagnts
From: brianf@dna.uvm.edu (Brain Foley)
Subject: Re: Re: 3' overlaps in PCR
Organization: Division of EMBA, University of Vermont
Distribution: bionet
Date: Wed, 16 Dec 1992 16:28:17 GMT

In my experience a three-base comlimentarity 5'-GAC-3' on one and
5'-GTC-3' on the other lead to complete failure to get the desired PCR
product.  I got all primer-dimer.  With a two-base complimentarity I
was able to get some product if the initial template concentration was
high enough.

Although 3 bases of anealing does not seem like much, consider the
high concentraion of primer relative to template DNA in the initial
rounds. And consider that once a single primer-dimer is formed it acts
as a template with 100% complimentarity to BOTH primers.

I say avoid 3' complimentarity at all costs!
--
********************************************************************
*  Brian Foley               *     If we knew what we were doing   *
*  Molecular Genetics Dept.  *     it wouldn't be called research  *
*  University of Vermont     *                                     *
********************************************************************
From: greenleaf@bchm.biochem.duke.edu
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Q: fusion protein expressin
Date: 18 Dec 92 22:10:47 GMT
Reply-To: Andrea Skantar
Distribution: usa
Organization: biochem

In article <1992Dec17.052937.12926@nuscc.nus.sg> mcbzzs@nuscc.nus.sg (Zhao
Zhuoshen (Mr)) writes:
>Dear fellow-netters,
I normally collect 8 fractions 1ml each. But I can
>not get any proteins by this protocol. Are there any of you know what is
>wrong I did? Is there any people has a better protein expression and
>purification method? Any suggestion is appreciated.
>
>Thanks in advance.
>Jackson
>mcbzzs@nuscc.nus.sg
>
Your fusion protein is probably insoluble, so when you spin down the lysate,
what you want is in the pellet, in inclusion bodies.  You may have to resuspend
your inclusion bodies in an denaturant (see references by F.A.O. Marston for
ideas) to solubilize your fusion protein, then get rid of the denaturant.  I do
mine in 8M urea/50mM tris ph 8/ 50mM KCl/1mM edta/ 50mM DTT (to reduce
disulfides), homogenize in this, incubate for 1hr at 4degC, then dialyze into
avarious buffers without any salt (your protein may vary).  Then try the column
again.  My fusion still does not bind well to GSH agarose after this treatment,
but you may have better luck.  Hope this helps.


Andrea Skantar
Duke University Biochemistry Dept.
greenleaf@bchm.biochem.duke.edu 
Newsgroups: bionet.molbio.methds-reagnts
From: zsha@iastate.edu (Zhengyu Sha)
Subject: low Tm primer PCR
Organization: Iowa State University, Ames IA
Date: Mon, 14 Dec 1992 00:42:25 GMT

I have problem with a low Tm(46.8) 18mer primer in my PCR. My setting is:
95C 15sec.  41C 1.5 min. 72C 2.5 min. The other primer(18bp) has a 55.5 Tm. 
So the two primers are quite deferent in their Tm.( I know I should use two
two primer which have the same Tm above 55C, but I just want to use the primers which I synthesized for sequencing). I have no problem when i use a primer(Tm 48.6) with annealing Tm=43C in my PCR (the other primer is that 55.5 Tm primer). My question is since the annealing Tm is quite low do I have to low my extension Tm( I tried 70C, no help)?
Thanks in advance!  
-- 
 $====================================|=======================================$
 $       Zhengyu Sha                  |    2188 Molecular Biology Building    $
 $   Phone No. (515)294-4210          |    ISU, Ames,Iowa 50011               $
 $=I think what I said is the official opinion of ISU, but no one believe it.=$
From: vrati@dbe.csiro.au (Sudhanshu Vrati)
Newsgroups: bionet.molbio.methds-reagnts
Subject: protein seq. alignment programe
Date: 18 Dec 92 21:11:55 GMT
Distribution: bionet

I am trying to align more than three protein sequences using gcg pile up
programe.  Are there any other programes available for this purpose ?
Thanks for your response,
Sudhanshu

********************************************************************************
    Sudhanshu Vrati, PhD                                  Tel: 61-2-8864964
    CSIRO Division of Biomolecular Engg.,                 Fax: 61-2-8864805
    PO Box 184, North Ryde, NSW 2113              Email: vrati@dbe.csiro.au
    Sydney (Australia) 
********************************************************************************
From: mschweder@gnv.ifas.ufl.edu (Mark Schweder)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Agarose gel migration artifacts of PCR products.
Date: 11 Dec 92 20:47:32 -0500
Distribution: bionet
Organization: Agronomy Dept, Univ of Florida

In article <9212100007.AA26748@phobos.med.pitt.edu>, bsh@MED.PITT.EDU (Basavaraju Shankarappa) writes:
> 
> Hi Netfolks:
> 	We have been trying to resolve the amplified product on a
> agarose gel.  Usually we have no problem.  But in one case we tried
> primers that contained lot of degeneracies.  When we run this product
> on an agarose gel, there is a kind of smearing effect in the lanes.
> This smear is clearly not caused by the presence of products of varied
> sizes.  The lane looks like the DNA appears to have aggregated and
> travels as a streak along the two edges of the lane.  We can also 
> faintly see the band at the appropriate positions.
> 	We tried running the amplified product after diluting it and 
> also added SDS.  But it does not seem to go away.  Has anybody else
> seen this happen or has worked out a solution.  If I get a reply
> that solves the problem, I will post on the net.
> 	Thanks 
> Raj Shankarappa
> Univ of Pittsburgh
> bsh@med.pitt.edu

 Try using a 3% gel that is composed of 50% normal agarose (SeaKem) and 50% low 
melting point agarose (NuSieve). It will increase resolution tremendously, but 
probably won't get rid of a long smear.
-- 
   ---------------------------------------------------------------------------
   Mark Schweder                | There are three things that smell like fish!
   Plant Science Laboratory     | One of them is fish...
   Agronomy Department          | The other two...
   University of Florida        | Are growing on you...
   Gainesville, Florida 32611   | (Frank Zappa, Jumbo Go Away)
   ---------------------------------------------------------------------------
           904-392-1890         |         mschweder@gnv.ifas.ufl.edu
   ---------------------------------------------------------------------------
                  DISCLAIMER: I know nothing about nothing.
Newsgroups: bionet.molbio.methds-reagnts
From: pnh@fcsparc6.ncifcrf.gov (Paul N Hengen)
Subject: Re: DNA from Polyacrylamide Gels
Summary: micro-elution systems (CHEAP!)
Organization: Frederick Cancer Research and Development Center
Distribution: bionet
Date: Fri, 18 Dec 1992 18:04:21 GMT

Basavaraju Shankarappa (bsh@MED.PITT.EDU) wrote:

> I was wondering if anyone out there knows of any company that sells a kit
> for extracting DNA from polyacrylamide gels.

    -- stuff deleted --

Then, C. Victor JONGENEEL (vjongene@isrecmail.unil.ch) wrote:

>Another (and easier) way is to electro-elute your DNA from the gel.
>There are several commercial systems available to do this....[etc.]

If you don't want to buy the very expensive unit for electroelution,
you might try using a few odds and ends around the lab. For example,
this could easily be done using a microtitre dish and a small cappillary
tube. Heat the cappillary tube and bend it to form a U-like structure.
Fill the tube with electrophoresis buffer using a micropipettor. Then,
place the tube so that it spans two of the titre dish wells. This can
be done in an array so that several can be done at the same time. Tape
a bent platinum wire into each well and connect your power supply. You
might consider testing this first with a dye solution. This is a simple
and CHEAP method that is a modification of those published:

@ARTICLE{Karuppiah1992,
AUTHOR = "N. Karuppiah
        and P. B. Kaufman",
TITLE = "Rapid and inexpensive micro-electroelution of nucleic
        acid and protein from agarose and polyacrylamide gels",
JOURNAL = "Biotechniques",
YEAR = "1992",
VOLUME = "13",
PAGES = "368"}

@ARTICLE{Sandhu1989, 
AUTHOR = "G. S. Sandhu
        and B. C. Kline",
TITLE = "Inexpensive micro-electroelution apparatus for
        extracting DNA from acrylamide and agarose gels",
JOURNAL = "Biotechniques", 
YEAR = "1989", 
VOLUME = "7", 
PAGES = "822-823"}

Paul N. Hengen
National Cancer Institute
Frederick Cancer Research and Development Center
Frederick, Maryland 21702-1201 USA

Newsgroups: bionet.molbio.methds-reagnts
From: broe@aardvark.ucs.uoknor.edu (Bruce Roe)
Subject: Re: Acid washed sand
Originator: usenet@midway.ecn.uoknor.edu
Date: Fri, 18 Dec 1992 12:36:00 GMT
News-Software: VAX/VMS VNEWS 1.41    
Organization: University of Oklahoma - University Computing Services

In article <92352.133614LAKSHMAN@MAINE.MAINE.EDU>, LAKSHMAN@MAINE.MAINE.EDU writes...
>We are trying to isolate nuclei from a fungus (R. Solani)
>using a protocal which calls for "acid washed sand" to grind the
>mycelium. Does anyone know how to acid wash sand (we are trying 50%
>sulfuric acid overnight, is this appropriate)? Also why does it
>need to be acid washed? Perhaps to remove any metal ions?
> 
>Thanks,
>Dilip
>DILIP LAKSHMAN                             LAKSHMAN@MAINE.MAINE.EDU
>DEPT. PLANT BIOLOGY & PATHOLOGY
>UNIV. MAINE AT ORONO
>ORONO ME 04469

Dilip,
	It's along time ago that I did that, but as I recall, I just
used concentrated HCl directly out of the bottle. Let it sit for 15 min
and then rince extensively with distilled water.  Acid washing the sand
will remove organic material that might be contaminating the sand. 
	Cheers........bruce
  - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
 \  Bruce A. Roe               Professor of Chemistry and Biochemistry /
 /  University of Oklahoma     INTERNET: BROE@aardvark.ucs.uoknor.edu  \
  - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -  - - -