HDTenEightyP/NetNews huggingface dataset:
From: ca566@cleveland.Freenet.Edu (Michael Holloway)
Subject: Re: Two Sequencing questions:
Reply-To: ca566@cleveland.Freenet.Edu (Michael Holloway)
Organization: Case Western Reserve University, Cleveland, OH (USA)
Date: Tue, 18 Aug 92 23:43:37 GMT


In a previous article, ca566@cleveland.Freenet.Edu (Michael Holloway) says:

>I think they mean 33-P.  Call Amersham and ask them for information and a
>free sample.  It works as advertised.  You'll like it.  There was an
>article in Biotechniques about it 11(1):36.
>-- 
Sorry.  That's NEN (as someone else as already stated) not Amersham.

-- 
E-mail: mhollowa@ccmail.sunysb.edu (mail to freenet is forwarded)
phone:  (516)444-3612
I would claim to speak for my university but no one is likely to
believe it.
From: jdboer@SOL.UVIC.CA (Johan deBoer)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Automated sequencing
Date: 19 Aug 92 20:48:44 GMT
Distribution: bionet

Hello out there,
Automated DNA sequencers are used for sequencing DNA, and can also be
used for say determining the size of labeled DNA fragments.
I was wondering if anyone is aware of other possible or potential
applications of these machines. Any ideas?

Johan de Boer 
jdboer@sol.uvic.ca
From: txt15@po.CWRU.Edu (Tao Tao)
Newsgroups: bionet.molbio.methds-reagnts
Subject: pYZ4 sequence needed...
Date: 14 Aug 92 16:50:34 GMT
Organization: Case Western Reserve University, Cleveland, Ohio, (USA)


I wonder whether the sequence of plasmid vector pYZ4 is available or not.
If it is out there, could some kind soul on the net land forward a copy to
me.

Thanks in advance.

Tao
Newsgroups: bionet.molbio.methds-reagnts
From: ca566@cleveland.Freenet.Edu (Michael Holloway)
Subject: Re: Two Sequencing questions:
Reply-To: ca566@cleveland.Freenet.Edu (Michael Holloway)
Organization: Case Western Reserve University, Cleveland, OH (USA)
Date: Tue, 18 Aug 92 00:12:00 GMT


In a previous article, zsha@iastate.edu (Zhengyu Sha) says:

>In <1992Aug10.154723.23852@magnus.acs.ohio-state.edu> rrumpf@magnus.acs.ohio-state.edu (Robert Rumpf) writes:
>
>>I have two questions regarding sequencing protocols:
>
>>2.  Anyone have any experience with 33-P yet?  We would like to know if the 

>	You mean 32-P? The answer is yes. Even with 35-S sequencing gel we exposure the dried gel without removing the saran wrap. The results are satisfied if you labeling reaction is good. 
>	

I think they mean 33-P.  Call Amersham and ask them for information and a
free sample.  It works as advertised.  You'll like it.  There was an
article in Biotechniques about it 11(1):36.
-- 
E-mail: mhollowa@ccmail.sunysb.edu (mail to freenet is forwarded)
phone:  (516)444-3612
I would claim to speak for my university but no one is likely to
believe it.
Newsgroups: bionet.molbio.methds-reagnts
From: browns@ccu.umanitoba.ca (Stuart Brown)
Subject: Re: polysaccharides in DNA extractions
Organization: University of Manitoba, Winnipeg, Manitoba, Canada
Date: Fri, 21 Aug 1992 15:43:46 GMT

In article <1992Aug19.173942.28680@news.miami.edu> kramer@oj.rsmas.miami.edu (Jack Kramer) writes:
>
>DNA extractions for cacoa ( and some other tropical plants) are
>extremely difficult due to the co precipitation of mucus with the
>DNA.  I would like to avoid any gradient centrifugation so I am
>looking for a way to chemically or enzymatically digest the poly-
>saccharide without damaging the DNA.  Since DNA is also a 
>substituted polysaccharide, is this still possible?
>
>Thanks to all who clarified the identity and sources of Polyclar.
>It is the acid washed, insoluable (high molecular weight) form
>of polyvinylpolypyyrolidone.

I use a CTAB extraction to remove polysaccharides from plant and fungal DNA
preps.  Add CTAB to 1% and NaCl to 0.7M, mix, then extract 2X with chloroform.
Excellent description of this method in "Current Protocols in Molecualr Biology"(the Big Red Book).  I think removing the polysaccs. is much easier than trying
to digest them away.

	-Stu

-- 
Stuart M. Brown                             If you can remain cool when all 
U. of Manitoba, Dept. Plant Science         Around you are in panic,
Winnipeg, Manitoba, CANADA
browns@ccu.umanitoba.ca            Then you surely misunderstand the situation
From: bertheau@inapv.inapg.inra.fr (Yves Bertheau)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: polyclar AT
Date: 19 Aug 92 12:38:51 GMT
Organization: Institut National de la Recherche Agronomique INA P-G

In article <1992Aug19.003937.28881@news.miami.edu> kramer@umigw.miami.edu (Jack Kramer) writes:
>I have seen references to "Polyclar AT" for use in cleaning up 
>plant DNA extractions.  I can't find it anywhere.  Does anyone know of
>a vendor or another name for the reagent?

Look for Polyvinyl polypyrolidone
-- 
		Yves Bertheau
INRA INA P-G		Pathologie Vegetale	16 rue Claude Bernard	
75231 PARIS cedex 05	FRANCE	Tel +33 (1) 43.31.93.97 
Fax: +33 (1) 43.31.83.82 Internet: bertheau@inapg.inra.fr
Newsgroups: bionet.molbio.methds-reagnts
From: robert@arbo.microbiol.uwa.oz.au (Robert Coelen)
Subject: Re: Two Sequencing questions:
Organization: University of Western Australia
Date: Thu, 13 Aug 1992 03:53:28 GMT

>In <1992Aug10.154723.23852@magnus.acs.ohio-state.edu> rrumpf@magnus.acs.ohio-state.edu (Robert Rumpf) writes:
>
>I have two questions regarding sequencing protocols:
>
>1.  Our 35-S gels are still sticky after drying; the saran wrap sticks to them 
>and is difficult to remove.  Why?  We also noticed that removing the saran wrap
>and placing film directly on the gels apparently causes some damage to the 
>film from the moisture or urea in the gel...
This is a frequently recurring theme. In our lab we don't remove the saran wrap
All we do is dry our gels on a vacuum dryer with the heat turned up to 80 dgc, leave
the wrap and expose overnight to good effect. If your signal is a bit weak,
examine your template (is it clean, is there enough ?) and the template to
primer ratio.

>
>2.  Anyone have any experience with 33-P yet?  We would like to know if the 
>energy from this isotope is sufficient to pass through saran wrap so we can 
>expose films without taking the wrap off.

We'll post an answer to Q2 tomorrow (it's running right now !)

Regards,

Robert (Semi-mature Gouda CHEESE) Coelen
Dept of Microbiology
UWA
From: bsh@MED.PITT.EDU (Basavaraju Shankarappa)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: InVitroGen Dipsticks?
Date: 19 Aug 92 18:52:56 GMT
Distribution: bionet

> >Any netheads out there have any experience with the InVitroGen Dipsticks used 
> >for determining DNA quantity in solutions?  I have heard the literature
> claims
> >of being as easy to use as litmus paper, sensitive enough to detect 20ng/ul 
> >with only a 1ul sample, etc.  What I would like are personal 
> >experiences/opinions, etc on whether this stuff works, is cost-effective, 
> 
> G'day all,
> 
> To anyone thinking of buying the DNA Dipstick I'd suggest that, unless
> they have improved their product in the last year, you'd be better off
> running your sample along with some standards on a mini agarose gel for
> 30min - in my hands this gives a more accurate estimation of DNA
> concentration and also leaves you free for 30 minutes to do other things
> (like read Bionet News :)
> 
> Bill Warren
> 
> 
I basically agree with this observation.  The KIT is a BAD kit.
I have not been able to get quantitation of DNA to any reasonable accuracy
using this kit and I have also decided that it is a worthless product.
Raj Shankarappa

Newsgroups: bionet.molbio.methds-reagnts
From: mcao@vthvax.tamu.edu
Subject: pXM vector sequence
Organization: Veterinary Teaching Hospital, Texas A&M University
Date: Thu, 13 Aug 1992 16:53:10 GMT

	Hi, Does anyone in this group have COS-1 cell expression vector pXM
sequence and kindly e-mail to me? Thanks.

					Ming Cao
Newsgroups: bionet.molbio.methds-reagnts
From: ca566@cleveland.Freenet.Edu (Michael Holloway)
Subject: Re: Lab stocks database: re-re-re-inventing the wheel
Reply-To: ca566@cleveland.Freenet.Edu (Michael Holloway)
Organization: Case Western Reserve University, Cleveland, OH (USA)
Date: Sun, 16 Aug 92 02:26:31 GMT


In a previous article, dadler@u.washington.edu (David A. Adler) says:

>around. The current requester has a DOS machine and RBASE and was quoted  
>$6,000. to have her freezer stocks' database created.

Typical.  I apoligize to you hard working software developers out there,
but these people seem to have no concept at all about what kind of budgets
we work with.  I've had salespeople tell me that all we have to do is go
off and write a grant for the money.  It's like  we're lazy because we
can't pay their price.  It never seems to occur to anyone that they have to
make a product that the typical lab can afford.

>THE IMMEDIATE QUESTION: Does anyone have a sample stock database application  
>for RBASE that they would be willing to share?

Spreadsheet or simple flat file database programs are more than adequate to
the task, or so I naively assume.  Can anyone explain why not?

>THE BIGGER QUESTION: Should someone, let's say NIH, fund some lab management  
>database development so that duplication and disaster can be avoided?
>(What's $6,000 x the # of labs that need such software?)

This argument never got very far to saving Bionet, did it?  Think of all
the wasted money from people duplicating database and analysis facilities,
not just institution to institution but in lab to lab, sometimes in labs
right next door to one another!

Mike

-- 
E-mail: mhollowa@ccmail.sunysb.edu (mail to freenet is forwarded)
phone:  (516)444-3612
I would claim to speak for my university but no one is likely to
believe it.
From: lpan@uk.ac.crc (Dr. Langxing Pan)
Newsgroups: bionet.molbio.methds-reagnts
Subject: application
Date: 21 Aug 92 17:55:06 GMT
Distribution: bionet
Original-To: methods@uk.ac.daresbury

Can I join the list?

Dr. Pan LX
Histopathology
UCL
London
Newsgroups: bionet.molbio.methds-reagnts
From: gat44@iastate.edu (Gabriel Trueba)
Subject: suscription
Organization: Iowa State University, Ames, IA
Date: Fri, 14 Aug 1992 01:50:54 GMT

 I would like to suscribe

Gabriel Trueba
Iowa State University
gat44@iastate.edu
From: naffara@crc.ac.uk (Dr. N.A. Affara)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Screening cDNA Libraries
Date: 13 Aug 92 12:02:50 GMT
Organization: MRC Human Genome Resource Centre

In article <1992Aug12.215726.1648@ucbeh.san.uc.edu>, penas@ucbeh.san.uc.edu writes:
> Hello!
> 
> I am soon going to start screening a lambda gt10 cDNA library.  This has never
> been done successfully in my lab. 

(rest of article deleted)

> Sandra Pena de Ortiz     : penas@ucbeh.bitnet
> University of Cincinnati
> College of Medicine
> Toxicology Program

I am also interested in screening a gt10 cDNA library.
Are there any ways which _don't_ use radioactivity?
Please mail any suggestions to me. (or post if you must ;->)

Phil
(E-mail naffara@uk.ac.crc)

-- 
Kryten (When Cat says why don't we raise the defensive shields?) "A superlative
suggestion sir,with only two drawbacks: one,we don't have any defensive shields
and two,we don't have any defensive shields.I know that,technically,that's only
one drawback,but it was such a big one I thought I'd mention it twice".
Newsgroups: bionet.molbio.methds-reagnts
From: ca566@cleveland.Freenet.Edu (Michael Holloway)
Subject: Re: acquisition of sequence from genbank
Reply-To: ca566@cleveland.Freenet.Edu (Michael Holloway)
Organization: Case Western Reserve University, Cleveland, OH (USA)
Date: Tue, 18 Aug 92 23:19:01 GMT


In a previous article, bsh@MED.PITT.EDU (Basavareju Shankarappa) says:

>Here is what you can do to directly get a sequence from genbank:
>
>send a mail message to retrieve@genbank.bio.net
>with nothing in the subject. And in the body of the message write
>only
>the accession number ( or locus name).  If you have more than one
>accession
>numbers, write one below the other, one on each line; nothing else. 
>You will
>get the sequence.
>

You can also telnet to genbank.bio.net or genbank.ig.com maintained by 
Intelligenetics.  They'll give you 10 or 15 minutes on-line.   
 login: genbank, password:4nigms	
You'll be connected to an IRX interface that will search a number of
sequence databases as well as the new data.

Does anyone know what's going to happen to this service when Genbank moves?

Mike
-- 
E-mail: mhollowa@ccmail.sunysb.edu (mail to freenet is forwarded)
phone:  (516)444-3612
I would claim to speak for my university but no one is likely to
believe it.
From: DMEIER@mis.mcw.edu
Newsgroups: bionet.molbio.methds-reagnts
Subject: Immunoprecipitation
Date: 12 Aug 92 21:36:14 GMT
Distribution: bionet
Organization: Medical College of Wisconsin (Milwaukee, WI)

Hello all,  I'd appreciate your advice.  What is the best way to
immunoprecipitate protein now-a-days?  My strategy is: (1) check homogenate
for protein using Western, (2) immunoprecipitate protein and show 
(a) the protein is in the pellet and not in the supernatant using Western
blotting.  My antibody is raised in rabbit.  Is Staph A best? What about 
antirabbit IgG/Sepharose?  Thanks much.

Dan Meier
From: bsh@MED.PITT.EDU (Basavaraju Shankarappa)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: TMACl hybridization of RNA:DNA
Date: 19 Aug 92 13:22:17 GMT
Distribution: bionet

> Furthermore I would like if somebody has some experience on how to keep the
> DYNA-beads from falling to the bottom (I know the hard way, resuspending them
> mannully ones in a while, but as a normal human beeing, I am seeking for some-
> thing to do the job for me)
> Peter Kristensen
> Department of chemistry
> Division of Biostructural Chemistry
> University of Aarhus
> Denmark
> 
> e-mail : Brian@bio.aau.dk

I use a tube rotator to keep things mixed up (!) all the time.
I guess you can have one home-made by connecting a low speed motor
to a round plate.  If you can place the tubes on the plate and have it
rotating perpendicular to the table surface.  
This way the contents will be continously inverted ensuring an efficient
mixing.  
This comes very handy for me for a GENTLER AND KINDER phenol extraction 
where I cannot do vortexing.  
For about US$ 100 you may be able buy it from hospital supplies.  
What I have is made by
Scientific Equipment Products
2201 Aisquith St
Baltimore MD 21218
\

Raj Shankarappa
Univer of Pittsburgh.
From: DMEIER@mis.mcw.edu
Newsgroups: bionet.molbio.methds-reagnts
Subject: Protein microsequencing
Date: 19 Aug 92 00:59:14 GMT
Distribution: bionet
Organization: Medical College of Wisconsin (Milwaukee, WI)

Hello All,
  To sequence the amino terminal end of a peptide, a facility in Wisconsin 
requires 150 pmol of protein. If the protein is approx. 60,000 daltons, 
then sequencing requires 3 micrograms of pure protein.  Is that typical 
for protein sequencing facilities?  I'd appreciate your replies.

Dan Meier
FR*ATLAS*CICT; arrival Tue, 18 Aug 92 12: 29:40 +0100 action Relayed
From: CAMPBELL@EARN.FRCICT81
Newsgroups: bionet.molbio.methds-reagnts
Subject: home-made PCR gems reprise
Date: 18 Aug 92 11:29:40 GMT
Distribution: bionet
P1-Message-Id: FR*ATLAS*CICT; 920818122428839-CICT-TLSE
Original-To: methods@UK.AC.DARESBURY
Ua-Content-Id: 920818122428839-

Fellow netters,
A while ago someone posted a notice which stated that they used beads of
embedding paraffin to make home-made PCR gems.  Unfortunately, I inadvertently
deleted this posting and was wondering if the original author could send me
a protocol for making PCR gems from embedding paraffin.  Also, unlike the
original 'poster', I have only a solid piece of embedding paraffin and
was also wondering if anyone had any experience with similar material (ie.
not beads).  Presently I melt the sterile paraffin and then drop 50 uL
aliquots onto a sheet of Parafilm.  I pick the cooled 'gems' off of the
Parafilm and drop them on top of my PCR reaction (the fit isn't so good but
the drop melts immediately during my hot-start anyway).  After my reactions
are finished, I poke my pipette tip through the wax to obtain an aliquot
of the reaction.  Questions: Am I doing things right?  Is it possible to
just drop hot paraffin on top of my reactions and thus bypass the 'gem
synthesis' step?  Should I melt the paraffin at 72 C after my reactions
are finished when I want to obtain an aliquot of reaction product?
 
Pardon the naivete of my questions but I've never used (nor has anyone
around here) the original AmpliWax (TM) PCR Gems and I set up my 'gems'
based on what seemed to be the great principle which, unfortunately, used
very expensive hunks of wax.
 
Thanks,
 
Malcolm
 
Malcolm M. Campbell, Ph.D.
Equipe Lignines
Centre de Physiologie Vegetale
Toulouse, FRANCE
CAMPBELL@FRCICT81.BITNET
 
Disclaimer:  Malcolm qui?
From: TY9283@UACSC2.ALBANY.EDU
Newsgroups: bionet.molbio.methds-reagnts
Subject: EMail address
Date: 19 Aug 92 21:37:00 GMT
Distribution: bionet

Hi,

   Does anyone out there know the EMail address for any of the following
people?   They are all from University of Michigan Medical School.

   Brent W. Weston
   Rajan P. Nair
   Robert D. Larsen
   John B. Lowe

                                                        Thanks

                                                            J.W.
From: WAGNERF@BRUNB.BITNET (Wagner Fontes)
Newsgroups: bionet.molbio.methds-reagnts
Subject: RE: Protein Microsequencing
Date: 21 Aug 92 15:33:49 GMT
Distribution: bionet



      100 - 150 pMol is a standard for most protein sequencing centers,
   and the reason for that is to increase the probability of obtaining
   a N-terminal sequence as long as possible.

      If your protein is very hard to obtain, and you do not need more
   than 10 residues from the N-terminal, it is possible to try sequencing
   at the 1 pMol range.



                        Dr. Wagner Fontes
                        Brasilian Center of Protein Sequencing
                        Biochemistry-Protein Chemistry Laboratory
                        University of Brasilia
                        Brazil
From: DN
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: InVitroGen Dipsticks?
Date: 20 Aug 92 15:44:00 GMT
Organization: University at Buffalo
News-Software: VAX/VMS VNEWS 1.41

In article <1992Aug19.171937.10562@magnus.acs.ohio-state.edu>, rrumpf@magnus.acs.ohio-state.edu (Robert Rumpf) writes...
>Any netheads out there have any experience with the InVitroGen Dipsticks used 
>for determining DNA quantity in solutions?  I have heard the literature claims 
>of being as easy to use as litmus paper, sensitive enough to detect 20ng/ul 
>with only a 1ul sample, etc.  What I would like are personal 
>experiences/opinions, etc on whether this stuff works, is cost-effective, 
>etc...
> 
>Bob

A colleague has tried the Dipstick, and says that it works. However, it
requires some processing of the stick before quantitation. Also, though the
ad does not state it, the kit instructions note that it detects not only
DNA, but RNA as well. If working with Ethidium Bromide holds no fear for
you, you might try a fluorescence spot test we often use. Briefly, 2 ul
droplet of the DNA whose concentration is spotted on saran wrap layed on a 
UV transilluminator. An 8 ul droplet of a .5 ug/ml solution of EtBr is mixed
by pipetting up-and-down. A series of droplets containing known quantities
of DNA are also spotted. The fluorescence of the test sample is then compared
to the knowns. Like the Dipstick, the best response is obtained with amounts
as low as 1 ng an as high as 10 ng. A picture can taken with the appropriate
filter for a permanent record (and also to avoid having to stare at a UV box).
When quantitating, be sure to include a zero ng control, since the EtBr itself
can fluoresce. Also, ignore the fluorescence seen at the edges of the sample.
Once the solutions of EtBr and the known samples are prepared, this method
takes only a few minutes to do.

DN
From: P_TRAVERS@UK.AC.ICRF (PAUL TRAVERS)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Wanted - Kd transfected into RMAS or T2
Date: 19 Aug 92 12:40:00 GMT
Distribution: bionet
Original-To: METHODS@UK.AC.DARESBURY

Dear Netfolk,
	Does anyone have, or know of, transfectants of Kd into RMAs or T2, or any
TAP- recipient.  I would like to screen some peptides for binding to Kd.

		Paul Travers
ICRF Structural Molecular Biology Unit
Birkbeck College
Malet Street
London WC1E 7HX

P_TRAVERS@ICRF.ICNET.UK (Internet)
P_TRAVERS@UK.AC.ICRF 	(JANET)
Newsgroups: bionet.molbio.methds-reagnts
From: txt15@po.CWRU.Edu (Tao Tao)
Subject: Antibody for lacZ-alpha??
Organization: Case Western Reserve University, Cleveland, Ohio, (USA)
Date: Fri, 21 Aug 92 18:02:04 GMT


Dear netters,

I would like to know from you, the informed sources, whether the antibody
(polyclonal or monoclonal) against lacZ alpha fragment is available.

I am think to use is to detect the fusion proteins with the lacZ alpha at the
C-terminus.

TIA for your info.

Tao
From: REE700A@MAINE.MAINE.EDU
Newsgroups: bionet.molbio.methds-reagnts
Subject: Peltier PCR Instruments - Possible explaination of mixed reviews
Date: 18 Aug 92 14:54:14 GMT
Organization: University of Maine System


  Recently Dr. Richard Levenson placed a query for suggestions on PCR
Instruments.  One person loved the MJ research Peltier-based system.
Another (Phil Carl of UNC) hated Peltier in general.  Another replied
with a summary of their own survey (lost name @ vtvm1...) with 12 of
15 suggesting the MJ Research PTC-100.

  So, I called MJ Research (800-729-2165) and asked the rep (Jim Michas)
why Peltier-based PCR instruments would have such mixed reviews.  He said
that their original machines (pre- July 1990) usedf commercially available
Peltier devices and these devices couldn't stand up to the constant cycling.
They realized this and performed some research into improved Peltier cells.

  In July 1990, they introduced instruments based on their own Peltier
cells.  Since then 13 units have been returned out of 1900 shipped.  There,
I'm done quoting their sales pitch.  How many people that don't like Peltier
systems have their adverse experience on POST-July '90 MJ RESEARCH
instruments?
     Thanks...   Jeff Andle
                 BIODE, Inc. & University of Maine
                 because two checks are better than one!
From: wmelchior@NTBTOX.NCTR.FDA.GOV ("Bill Melchior, NCTR/FDA")
Newsgroups: bionet.molbio.methds-reagnts
Subject: Cs gradients (from CHEESE thread)
Date: 12 Aug 92 13:20:53 GMT
Reply-To: wmelchior@NTDOC.NCTR.FDA.GOV
Distribution: bionet

>I'm still trying to figure out why anyone still uses Cesium
>gradients. As far as I can tell, they add a couple of days and
>a lot of trouble for very little gain. Yet I still see a lot
>of labs doing them.

When I want VERY clean plasmid, I use the Cs/ethidium method of Garger
et al (BBRC 117, 835-842, 1983).  It takes one day -- I spin for 4 3/4 hr.
________________________________________________________________________________
The opinions stated are mine, not those of NCTR or its sponsoring organizations.

Bill (Cheesit) Melchior                      ||   "You have lawyers the way
National Center for Toxicological Research   ||    other people have mice."
Jefferson, AR  72079                         ||
(501) 543-7206                               ||   -Kenneth Duncan, English
                                             ||    Health & Safety Executive,
WMELCHIOR@NTDOC.NCTR.FDA.GOV                 ||    to US regulators