HDTenEightyP/NetNews huggingface dataset:
From: fgarbrec@POST.ITS.MCW.EDU (GARBRECHT)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Help: Panning
Date: 12 Dec 92 17:52:59 GMT
Distribution: bionet

I'm not sure exactly what you are asking here, but (as an immunologist),
I have some experiance in the past using monoclonal antibodies to pan
for various lymphoid cell populations.  In particular, I have panned
for CD3, CD4 and CD8 bearing lymphocytes.  In general, panning is a
picky procedure and is dependent upon a number of factors, including
quality of plastic used, density of cell surface determinants, purity and
affinity of the antibody used, etc.  We have pretty much switched
entirely to immunomagnetic bead cell selection procedures.
Fred Garbrecht
Medical College of Wisconsin
fgarbrec@post.its.mcw.edu

On 12 Dec 1992, JnP wrote:

> Date: 12 Dec 92 01:23:30 GMT
> From: JnP <pnj@stein.u.washington.edu>
> To: methods-and-reagents@net.bio.net
> Subject: Help: Panning
> 
> 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
> 


Xref: sparky bionet.molbio.methds-reagnts:2934 bionet.general:1991 bionet.immunology:201
Newsgroups: bionet.molbio.methds-reagnts,bionet.general,bionet.immunology
Subject: MHC T-cell epitopes
From: vladimir@wehi.edu.au
Date: 17 Dec 92 19:20:06 +1000
Organization: Walter & Eliza Hall Institute

Dear netters,

We would like biomedical researchers to submit for comparative
analysis amino acid sequences (epitopes) that bind to major
histocompatibility (MHC) molecules and are recognized by T cells.
Sequences from mice or human proteins are suitable. They may ha
been identified because they stimulate T cell lines or clones in
a MHC-restricted fashion or by sequencing after elution from
purified MHC molecules.

Thanks,

Vladimir Brusic,
The Walter and Eliza Hall Institute
Melbourne, Australia
From: MNAVARRE@ANDESCOL.bitnet
Newsgroups: bionet.molbio.methds-reagnts
Subject: signoff
Date: 16 Dec 92 20:59:54 GMT
Distribution: bionet
Organization: Universidad de Los Andes - Bogota, Colombia

signoff
Newsgroups: bionet.molbio.methds-reagnts
From: shecht@lamar.ColoState.EDU (steven heckt)
Subject: Re: low Tm primer PCR
Date: Tue, 15 Dec 1992 22:08:09 GMT
Organization: Colorado State University, Fort Collins, CO  80523

	You should not have to lower the extension temperature. In
	working with much lower Tm primers, you can consider the
	extension rate of your pol, which is slow but still going
	at lower temperatures, and sa you get extension the Tm will
	increase. So- you may consider using ramps; ie, ramp down to
	annealing slowly to allow the best matches and ramp up to
	your extension temp to allow the interplay of annealing and
	extension to work itself out.
Organization: University of Maine System
Date: Thursday, 17 Dec 1992 13:36:14 EST
From: <LAKSHMAN@MAINE.MAINE.EDU>
Newsgroups: bionet.molbio.methds-reagnts
Subject: Acid washed sand

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
From: DMEIER@mis.mcw.edu
Newsgroups: bionet.molbio.methds-reagnts
Subject: m rna extraction from tissue
Date: 11 Dec 92 21:03:38 GMT
Distribution: bionet
Organization: Medical College of Wisconsin (Milwaukee, WI)


 
Greetings,
     
We are having problems with the yeild and degradation of poly A Rna (via Fast
Track i.e, oligo cellulose ) after total RNA isolation from tissue. There are no problems using these techniques on cell culture material. We are working with gut and prostate tissue.

The yield of mRNA and the relative amounts of m RNA to total RNA is low. Does anyone have the same problem and how can these difficulties be overcome when extracting tissue?

Some one told us that there are high levels of endogenous RNAses in certain tissues. If this is true how can one overcome this problem?

Send replies to Dan Meier
DMEIER@mis.mcw.edu

Thank you,
Kathie Hopp and der O/TAN
From: HRMBDKC@HKUMD1.HKU.HK
Newsgroups: bionet.molbio.methds-reagnts
Subject: automatic DNA sequencing systems
Date: 18 Dec 92 07:19:00 GMT
Distribution: bionet

I'd like to get some feedback about how cost effective it is or desirable
it is to have an automatic DNA sequencer. How much sequencing would
be required to make it worth while to buy?
And does this really save time?
Is the sequence produced accurate?
Look forward to the responses!
Kathy Cheah
HRMBDKC@HKUCC.BITNET
From: ccy@po.CWRU.Edu (Cheung C. Yue)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: 5' RACE after RNA ligase - reference needed
Date: 11 Dec 1992 21:50:34 GMT
Organization: Case Western Reserve University, Cleveland, OH (USA)
Reply-To: ccy@po.CWRU.Edu (Cheung C. Yue)
NNTP-Posting-Host: thor.ins.cwru.edu


In a previous article, ccy@po.CWRU.Edu (Cheung C. Yue) says:

>
>Some time back I had come across a paper on 5'-RACE after: 
>1.  RNA ligase-mediated ligation of first strand cDNA (specifically
>    primed from internal sequence primer).
>2.  PCR using the internal sequence primer and its antisense partner.

Thanks to Vladimir (see enclosed message), an article, albeit not the
one I remembered, deals with this topic.  Since I cannot e-mail Vladimir,
I thought I'd thank him here, and to let everybody know about this reference.

ccy@po.cwru.edu
____

Received: by CSPGCS11 (Mailer R2.07) id 7425; Fri, 11 Dec 92 12:35:34 SET
Date:         Fri, 11 Dec 92 12:33:55 SET
From: "V.Korinek" <UMG17@CSPGCS11>
To: ccy@po.cwru.edu

 I know similar (but probably different) paper
published in Proc. Natl. Acad. Sci. USA, Vol.89
pp.9823-9825, October 1992: Ligation-anchored
PCR: A simple amplification technique with single-
-sided specifity.
 Maybe it could help you.
           With greetings from Prague
                                    Vladimir
--
From: cenderli@nyx.cs.du.edu (C. S. Enderlin)
Subject: Re: (none)
Organization: Nyx, Public Access Unix @ U. of Denver Math/CS dept.
Date: Sat, 12 Dec 92 04:49:32 GMT

In article <9212120019.AA08502@iastate.edu> pakapke@IASTATE.EDU writes:
>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.

A friend of mine electroporates single-stranded phagemid DNA (no phage
packaging) into E. coli using conditions worked out by the lab with
the electroporator for all their transformations.  The transformation
frequency with ssDNA is about 10E3 lower with ssDNA than dsDNA (with
standard transformation as well as for electroporation if I remember
correctly).  However, that probably isn't what you are asking about.
And, I don't know of any publications.

You're probably asking about lambda?  The only problem I see with that
is that most lambda ligations are set up to make concatamers (or 
using double cos sites?)
From: LCNAVOT@WEIZMANN.WEIZMANN.AC.IL (Nir Navot)
Newsgroups: bionet.molbio.methds-reagnts
Subject: PIPES
Date: 15 Dec 92 12:17:57 GMT
Distribution: bionet
Original-To: METHODS@uk.ac.daresbury

A friend wishes to prepare a 1M solution of PIPES (buffer) at pH 6.4. He needs
it for working with RNA. Questions: 1. What should be used for its titration?,
NaOH / KOH or else? 2. Can PIPES be autoclaved ? Should it be autoclaved at all
.... any reason to think there would be RNAases there? 3. Can it be autoclaved
in the presence of DEPC?

Thanks in advance,
Nir
From: afc@gnv.ifas.ufl.edu
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: colony screening with Amersham ECL (RPN 3000 and 3004)
Date: 17 Dec 92 14:12:57 GMT

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 !

This may not have anything to do with your problem, but since no one else
has responded:

We tried using various nonradioactive detection kits with probes hybridized
to squashed mosquitoes.  We got very high backgrounds with every one that
we tried.  We then tested just the reaction reagents with the squashes (no
probe).  Again, we got very high background.  Obvious conclusion: insects
have peroxidases and alkaline phosphatases that survive NaOH, baking, and
hybridization.  Maybe you have the same problem with bacteria, since colony
lifts are full of protein.

We were able to eliminate this background by treatment with protease.  We
used a laundry stain remover which is about 500000 times cheaper than 
proteinase K (your prices may vary).  Details are upcoming in a note by
Johnson et al. in BioTechniques in March.

Andrew Cockburn
USDA

> Christiane Bouchier
> Genethon
> 1, rue de l'Internationale
> 91 000 EVRY
> FRANCE
> Tel : 19.33.1.69.47.29.66
> email : bouchier@genethon.fr
Newsgroups: bionet.molbio.methds-reagnts
From: mcbzzs@nuscc.nus.sg (Zhao Zhuoshen (Mr))
Subject: Q: fusion protein expressin
Organization: National University of Singapore
Date: Thu, 17 Dec 1992 05:29:37 GMT

Dear fellow-netters,
I have had difficulty to express my gene(1.2kb) using GST-fusion protein
vector(P^GEX-2T) from pharmcia. What I did is as following:

1. construct the plasimd(P^GEX-2T with 1.2kb inframe insert), transform
it into E.coli(XL1-Blue strain) and choose the colony with 1.2kb insert.

2. grow up the bugs in 1XLB +amp medium o/n at 37C. add 10 ml the o/n
culture into 400 ml fresh 1XLB +amp,incubate at 37C for 4 hr.

3. Induce the fusion protein expression with 0.1mM IPTG and incubate it
at 37C for 3 hr. Then harvest the cells and resuspend the pellet with
15ml PBS+protease inhibitors. Sonicate and spin down it and pass the
supernatant through GST-sephorose column twice.

4.wash the column with PBS 4X before elute the fusion protein with 5mM
Glutathione(reduced). 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

Newsgroups: bionet.molbio.methds-reagnts
From: chisholm@.cmsbio.nwu.edu (Rex L. Chisholm)
Subject: Re: electroporation (to make cDNA libraries)
Organization: Northwestern University, Evanston, Illinois, USA
Date: Tue, 15 Dec 1992 14:00:48 GMT

djt2@po.CWRU.Edu (Dennis J. Templeton) writes:


>In a previous article, pakapke@IASTATE.EDU () says:

>>
>>     We would like to get away from in vitro packaging our phage DNA and
>>simply electroporate it into the cell directly.  Anyone in net-land had
>>experience doing this?  Are there any references published using this
>>technique to produce a phage library?
>>

>Hmm.  I don't have a direct answer for you, but I would be cautious about
>trying to electroporate DNA as large as the concatemers of lambda you get
>after ligation.

>On the other hand, we spent months making a good cDNA library out of
>LambdaZAP, THEN got experienced in electroporation of coli.  Given the high
>electrocompetency rate (10e10 is not unrealistic) it seems we might have
>been just as successful at making our cDNA library in pBluescript.

>Here are some numbers:

>5 ug A+ RNA, makes about 5 ug DS cDNA
>Ligating 1 ug of this into 0.5 ug of *prepared* (see below) pBluescript
>	should result in about 0.1 ug of circularized vector (probably
>	more)
>Electroporation of this (in several batches) has the potential to generate
>10e8 to 10e9 clones.  

>From a practical standpoint, the high electroporation efficiencies are at
>very low levels of supercoiled plasmid.  We have never gotten more than
>10e6 clones from a single pulse, but 10 pulses of a ligation are practical,
>and 10e6 is plenty of cDNA's anyway.

>Input from any who has made libraries this way would be appreciated, and
>those contemplating it should look at the article in this month's
>BioTechniques (13, Nr6. 862-864, 1992) wherein the authors describe using
>pBluescript to make T-tailed vector to clone cDNA's a al Okayama and Berg.

>good luck,

>dennis


We have been trying to do the same thing recently but have been unsuccessful
at achieving any significant level of introduction of lambda DNA, even though
plasmid gives us >10e9 transformants/microgram.  Anyone else have luck with
lambda DNA and electroporation?

Rex Chisholm
chisholm@worms.cmsbio.nwu.edu
Newsgroups: bionet.molbio.evolution
From: johnest@ulrik.uio.no (John E. Stacy)
Subject: Looking for info on mtDNA variation and stratagies for detection
Organization: University of Oslo
Date: Thu, 17 Dec 1992 10:26:54 GMT


I want to know if anyone out there has experience with (vertebrate) mtDNA -
In particular, how much intraspecific variation is there in the "D-loop"?
Regarding intraspecific variation, is it all at the level of single base 
changes, or are there length variations (i.e. detectable as PCR product 
length variants).

All comments and references greatly appreciated.
Newsgroups: bionet.molbio.evolution
From: AAT@UCSVAX.UCS.UMASS.EDU (Gus Trautweiler)
Subject: Re: Wings and evolution
Organization: University of Massachusetts
Date: Wed, 16 Dec 1992 01:33:26 GMT
X-News-Reader: VMS NEWS 1.24

In <gj169883.724367284@cunews> gj169883@alfred.carleton.ca writes:

:Q  I wonder, then, what mechanisms can be used to
:Q explain members such as these.
:Q 
:Q 

I think off hand that feathers evolved to keep early reptiles warm and that
insect wings evolved and temp. regulators.  Check Evolutionary Biology by
Futyma.


Gus Trautweiler (The Reporter Gene!) *%%*     *%%*       *%%*     *%%*       *%%
Plant & Soil Science: Biotechnology  |  | % % |  | %   % |  | % % |  | %   % | 
University of Massachusetts  |   %   |  |  %  |  |   %   |  |  %  |  |   %   |
AAT@UCC.UMASS.EDU   |     |  | %   % |  | % % |  | %   % |  | % % |  | %   % |
%*     *%%*      *%%*     *%%*       *%%*     *%%*       *%%*     *%%*       *%%
Newsgroups: bionet.molbio.evolution
Subject: Re: Looking for info on mtDNA variation and stratagies for detection
From: robison1@husc10.harvard.edu (Keith Robison)
Date: 17 Dec 92 19:59:29 GMT

johnest@ulrik.uio.no (John E. Stacy) writes:


>I want to know if anyone out there has experience with (vertebrate) mtDNA -
>In particular, how much intraspecific variation is there in the "D-loop"?
>Regarding intraspecific variation, is it all at the level of single base 
>changes, or are there length variations (i.e. detectable as PCR product 
>length variants).

>All comments and references greatly appreciated.

D-loop polymorphisms were used extensively by the late A.C. Wilson
and co-workers in their studies of human evolution, as well as many
other studies.  Some references are:

	PNAS 88:1597.  1991
	Nature 350: 467.  1991.
	Mol.Biol.Evol. 8:475.  1991.
	
You might also try looking in the subject indices of J.Mol.Evol and
Mol.Bio.& Evol year end issues under "D-loop" or "mitochond-".


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

robison@biosun.harvard.edu
From: HXIE@MACC.WISC.EDU (HUIWEN XIE)
Newsgroups: bionet.molbio.evolution
Subject: off
Date: 12 Dec 92 04:44:00 GMT
Distribution: bionet

Please sign me off. Thanks.
From: thomasb@SPIDER.ENTO.CSIRO.AU (Thomas Boyce)
Newsgroups: bionet.molbio.evolution
Subject: Wings and intermediate phenotypes
Date: 16 Dec 92 02:28:15 GMT
Distribution: bionet

The recent discussion by Ben Jones of the value of so-called
less than perfect phenotypes uses the evolution of flight as
an example where incremental changes towards a flight mechanism
can be incrementally more fit.  

While this may be true for big, furry animals, its probably not
true for insects.  The work of Joel Kingsolver and Mimi Cole has
shown in an elegant fashion that the incremental addition of 
length to pronotal lobes in proto-flying insects actually has
no beneficial areodynamic effects.  Thus, the insect would have 
no incremental gain in fitness via flight by incremental lengthening of
its proto-wings until they had become large enough to actually
affect its gliding ability. 

The flying-squirrel hypothesis for the evolution of insect flight
does not appear possible. 

 What could have happened, is selection for larger
and larger surfaces for thermodynamic control of body temperature
(large surfaces for basking) which were then coopted for flight
once they were large enough.

The lesson from this is that small, incremental changes in phenotype
toward an optimum may be the most obvious path, but might 
not be biomechanically or physiologicaly possible and may
have arisen through various
mechanisms at various times in its evolutionary development.

(Sorry, I can't lay my hands of the references to Cole and Kingsolver's
work at the moment.  I'll keep looking.)

Thomas Boyce
Entomology, CSIRO
Canberra, Australia
thomasb@spider.ento.csiro.au
From: jonesbb@BELOIT.EDU (Ben Jones)
Newsgroups: bionet.molbio.evolution
Subject: Re: Wings and evolution
Date: 15 Dec 92 01:48:45 GMT
Distribution: bionet

Colin Rowat writes:

>  I have a question regarding the evolution of any species with
>specialized members that would require many generations to serve a
>useful purpose.  A specific example is that of wings: clearly, for the
>first n generations, the "ancestors" of wings would not be
>evolutionarily adaptive but would rather be a hinderance until they
>became flight-able.  I wonder, then, what mechanisms can be used to
>explain members such as these.

If you can show that the ancestors of a specialized member would indeed
have been a hindrance, then you would have a valid question.  However, the
ancestors of wings are not a good example.  Flying squirrels do not have
wings, and it will probably be millions of years before they can fly like
bats (if they ever do).  However, the membranes between their front and
rear legs are clearly useful for gliding safely out of trees to avoid
danger or to break a fall, and I see no reason to think that smaller
membranes were EVER a hindrance rather than a help.

In addition, animals that jump or run fast probably will run faster or jump
farther if they are aerodynamic.  Animals that jump may also find it useful
to have "control surfaces" which allow them to control their attitude while
ballistic.

Creationists often cite the perfection of the eye as an example of
something that cannot be explained by evolution.  But you can start
removing the advanced features of the eye one by one and still have
something that's better than nothing, even if you end up with a simple
trace of light-sensitive chemicals.  Even single-celled plankton detect
light and rise and fall in the ocean depending on the time of day.



Ben Jones                  BioQUEST / Department of Biology
jonesbb@beloit.edu         Beloit College, Beloit, Wisconsin
From: andrewh@uniwa.uwa.edu.au (Andrew Hobbs)
Newsgroups: bionet.molbio.evolution
Subject: Re: Evolution of wings
Date: 18 Dec 1992 11:17:56 +0800
Organization: The University of Western Australia
NNTP-Posting-Host: uniwa.uwa.edu.au
X-Newsreader: Tin 1.1 PL5

Hi,

With regard to the evolution of wings.  Sometime back in Nature (Lewis,
Nature, 1978, 276, p565) there was a report of a single homeotic
mutation in drosophila which turned the halteres into apparently normal
wings.  Does anyone know whether they were completely functional and
has anyone studied their effect on the aerodynamics of flight of the
mutant form. Thus there is potential for very rapid changes in
morphology with rather small genetic changes.  I realize that this
mutation is homeotic and it is just inappropriately expressing a fully
evolved structure, but it seems to me that we shouldn't dismiss the
importance of sudden major changes in morphological characteristics.
And not just size but shape etc.

Andrew Hobbs
Biochemistry
University of Western Australia

Andrewh@uniwa.uwa.edu.au
From: kliman@mbcl.rutgers.edu
Newsgroups: bionet.molbio.evolution
Subject: Re: creation or evolution, spontanious or not ?
Date: 12 Dec 92 18:18:29 GMT
Distribution: bionet

In article <1992Dec10.091625.8218@athena.cs.uga.edu>, keck@athena.cs.uga.edu (Paul Keck) writes:
> If you're interested in this kind of stuff, check out the group talk.origins.
> Much discussion there.

Although I hope you're not suggesting that discussion of this topic be moved
from Bionet to talk.origins.  I think this is the best forum available for
scientific discussion regarding the origin of life.

Rich Kliman, Rutgers University
From: SATTLERG@SIMSC.SI.EDU
Newsgroups: bionet.molbio.evolution
Subject: Looking for info on mtDNA variation and strategies for detection
Date: 17 Dec 92 22:36:49 GMT
Distribution: bionet


   A good starting reference would be:
Wilkinson, G. S. and A. M. Chapman. 1991. Length and sequence variation in
evening bat D-loop mtDNA. Genetics 128:607-617.
Newsgroups: bionet.molbio.evolution
From: gj169883@alfred.carleton.ca ( gj student 169883)
Subject: Wings and evolution
Organization: Carleton University
Date: Mon, 14 Dec 1992 21:08:04 GMT

  I have a question regarding the evolution of any species with
specialized members that would require many generations to serve a
useful purpose.  A specific example is that of wings: clearly, for the
first n generations, the "ancestors" of wings would not be
evolutionarily adaptive but would rather be a hinderance until they
became flight-able.  I wonder, then, what mechanisms can be used to
explain members such as these.

  If you know of anyone who might be able to help me with this
question I'd appreciate being put in touch.

  Thanks for your help.

  Colin Rowat
  Department of Psychology
  Carleton University.
From: MCCAINKW@DUVM.OCS.DREXEL.EDU (Kate McCain)
Newsgroups: bionet.molbio.evolution
Subject: Wings & evolution
Date: 15 Dec 92 14:48:13 GMT
Distribution: bionet

For insights into this _particular_ example -- you might chase work by John
Ostrom (JH Ostrom). While I can't remember the exact references, I clearly
remember several lengthy review articles discussing his theories of the
development of feathers/wings/flight in small bipedal reptiles -- where
wings were (in their more terrestrial phase) insect-batting-down devices. His
illustrations look remarkably like the Northern Mockingbird.

Kate McCain                       "Bibliometrics R Us"
College of Information Studies
Drexel University
Philadelphia, PA 19104

mccainkw@duvm.ocs.drexel.edu