HDTenEightyP/NetNews huggingface dataset:
From: GRAZIAWD@SPLAVA.CC.PLATTSBURGH.EDU ("William D. Graziadei")
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: About NIH Templates for MAC Word....
Date: 15 Apr 92 18:23:00 GMT
Article-I.D.: splava.48160DEE00A00A2F
Distribution: bionet

First *kudos* to Dennis.

To convert MAC Word Templates to DOS Word or W4W Templates, do the following:

If you have access to a MAC, after unbinhex and unstuffing bring up the
file with MAC Word and then Save As Word for DOS. Use apple file exchanger
to move the file from MAC to a DOS disk.


   Regards,

      Bill 

   Question: What is remedial mitosis?

   Signature and answer follows:


         _____________________________________________________________________
        / William D. Graziadei, Ph.D. / In Vitro Cell Biology & Biotechnology/|
       /=============================/======================================/_/
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"Remedial mitosis is a math course for cells that can't multiply or divide."
Instructor: Dr. G. Factor; Sections: G1, G0 (Remedial), S, G2, M; Time: Varies
Newsgroups: bionet.molbio.methds-reagnts
From: txt15@po.CWRU.Edu (Tao Tao)
Subject: Re: Computerized gel documentation
Organization: Case Western Reserve University, Cleveland, Ohio, (USA)
Date: Sat, 11 Apr 92 18:09:30 GMT


If you use Mac, one option you have would be using the scanner and Aldus'
PhotoShop to scan and store your gel image.  The drawback is that it is
memory demanding, a gel may use up to 8 mb of disk space.

To get around the problem some compression utilities may be used.

Tao
From: brknig01@uctvax.uct.ac.za
Newsgroups: bionet.molbio.methds-reagnts
Subject: mtDNA extraction from EtOH preserved specimens
Summary: mtDNA extraction from ethanol preserved animal material
Keywords: mtDNA extraction procedures
Date: 8 Apr 92 13:46:08 GMT
Organization: University of Cape Town

Does anyone in the big wide world know of a procedure for the extraction and
isolation of mtDNA (mitochondrial DNA) from Ethanol preserved specimens? I do
not need TOTAL DNA prep procedures; specifically for mtDNA. 

Thanks in anticipation

Nigel Barker and Colleen "I love rhino's" O'Ryan.
Please reply to Colleen@chempath.uct.ac.za this is our usual email address.
Newsgroups: bionet.molbio.methds-reagnts
From: cbeall@magnus.acs.ohio-state.edu (Clifford J Beall)
Subject: Re: reuse of Dynabead-oligo(dT)?
Organization: The Ohio State University
Distribution: bionet
Date: Mon, 13 Apr 1992 17:10:23 GMT

In article <9204111220.AA18791@genbank.bio.net> yhsun%imb.dnet@LAS.AS.EDU.TW writes:
>Can the oligo(dT)-Dynabead be reused? i.e. Can the oligo(dT)-dynabead
>withstand NaOH wash? Is there a temperature limit for the elution step?
>
>Henry Sun

Dear Henry,
A post-doc in our laboratory has re-used the beads by washing 5 times
in 0.1 N NaOH and then 5 times in 0.02% azide in PBS pH 7.5.  This 
procedure worked for at least a half dozen uses.  I have no information
on the temperature limit beyond the recomended 65 degrees.
All the best,
Cliff Beall
Ohio State Biotechnology Center
From: rybicki@uctvax.uct.ac.za
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: single-stranded PCR
Date: 9 Apr 92 16:39:44 +0200
Organization: University of Cape Town

In article <30MAR199215501948@rigel.tamu.edu>, msw1633@rigel.tamu.edu (Mark S. Whitsitt, N5RJF) writes:
> In article <1992Mar30.142854.9212@news2.cis.umn.edu>, ernest@lenti.med.umn.edu (Ernest Retzel (1535 49118)) writes...
>> 
>>Why not put a biotin on one of your primers, and fish it out after the
>>PCR with magnetic beads?  This has been done for a variety of situations
>>already.
>> 
>>Ernest Retzel
>>Dept. of Microbiology
>>Univ. of Minnesota
> 
> How about a reference or two on that.  I am not sure about how to biotinylate 
> the primer, ie how many biotins per primer, how much does the biotin affect the 
> PCR reaction , and how do you determine how many/much of the magnetic particles 
> do you add per estimated biotin content?
> 

Basically, you ask whoever is doing your oligos to use "biotin-on
phosphoramidite" chemistry: this ensures you get a biotin wherever you want one
in the sequence, but one should place it at the 5' end, where the odds are it
will not interfere with the annealing or with the extension, and which means
you can fish it out with any avidin/streptavidin conjugate, whether on a
magnetic bead, or column material.  Refs: none offhand, but any oligo-maker or
other PCR freak should have a bundle.  Also any archiving programme / online
literature database should be able to pull out a ton.

Ebnjoy

Ed Rybicki
Dept Microbiol, Univ Cape Town
From: ljlj@quads.uchicago.edu (loren  joseph)
Newsgroups: bionet.molbio.methds-reagnts
Subject: freezer monitor alarms
Date: 12 Apr 92 15:08:46 GMT
Article-I.D.: midway.1992Apr12.150846.10853
Reply-To: ljlj@midway.uchicago.edu
Distribution: bionet.molbio.mthds-reagnts
Organization: University of Chicago Computing Organizations

I hope this is an acceptable place to post this message- it does

not deal with methods but does bear on reagent welfare. Can 

anyone offer do-it-yourself advice on assembling a temperature

monitor-alarm-autodialer system fro multiple freezers.  Some already

have monitors with audible alarms, so hookup with a dialer should

be simpler.  Others would need a temp. probe, hopefully with a thin

lead that can sneak past the door gasket Several supply house catalogues

advertise reasonably priced models that seem like they should work, but

I do not have a good feel for what kind of power-output, leads and so

forth are needed to connect an autodialer.  I can refer people to one company/distributor

which supplies a four freezer-1 dialer system for about $1200, which seems

a bit steep (except compared to the aggregate cost of enzymes/bacterial 

stocks...).  I will be happy to compile and post (?compost?)direct

responses (I'll also try to find out how to reduce linespacing with

this editor).  Thanks, Loren Joseph (U. Chicago)
Newsgroups: bionet.molbio.methds-reagnts
From: kramer@news.miami.edu (Jack Kramer)
Subject: Re: Bind silane protocols for seq gels
Organization: University of Miami, RSMAS
Date: Thu, 9 Apr 92 12:34:27 GMT

In article <1992Feb25.214601.21945@acsu.buffalo.edu> camlau@ubvmsb.cc.buffalo.edu (B.J.) writes:
>In article <1992Feb25.141537.935@uwovax.uwo.ca>, 37_527@uwovax.uwo.ca (Gregory A. Denomme) writes...
>>  I would like to obtain receipes for coating the outer plates for sequencing
>>gels using BDH's Silane A-174 as the 'bind Silane'.  I am having some trouble
>>at the point of separating the plates and maybe a new protocol would help.
>
>Our lab uses PAM to help gels unstick.  That's right, PAM, the vegetable
>spray shortening.  Seems to work wonders.  Just spray on, wipe down excess.
>

Has anyone tried this for gels for IEF of proteins?  Sure is a lot
cheaper than the commercial repel silanes.

Is there a similar alternative for the "bind" side plate?

jack kramer
Univ of Fla
Tropical Reearch and Education Center
kramer@molbio.med.miami.edu
From: e_schroder@RUMAC.UPR.CLU.EDU
Newsgroups: bionet.molbio.methds-reagnts
Subject: Source of natamycin
Date: 8 Apr 92 23:16:54 GMT
Distribution: bionet


   Does anyone know a source for the fungal inhibitor natamycin?
    We need the solid compound. We know is produced or distribuited
in Europe by Gist-Brocades, Deft, The Netherlands; but need ther address or
US distribuitor.
   Send personal E-Mail to
                      E_SCHRODER@RUMAC.UPR.CLU.EDU
 Thanks
From: skspoidn@susssys1.rdg.ac.uk (Mike Poidinger)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: DIGESTION OF PCR PRODUCTS
Date: 10 Apr 92 13:08:21 GMT
Distribution: bionet

In response to:

----- Begin Included Message -----

Message-Id: <9204091841.AA13249@genbank.bio.net>
From: falquet@ch.unige.sc2a
Subject: Re: DIGESTION OF PCR PRODUCTS
Date: 9 Apr 92 13:20:45 GMT
Status: R

In article <9204010949.AA16192@genbank.bio.net>, UDHA094@OAK.CC.KCL.AC.UK
 (MARCEL KUIPER) writes:
>
> Dear colleagues,
>
>
> I am trying to introduce restriction sites at the 5' and 3' end of PCR
> products, to ligate the digested product in a vector. Until now I've
> been unsuccesfull in doing so. At the 5' end I've introduced a KpnI site
> with 6 bp in front of it; at the 3' end I've introduced a BglII site with
> 2 bp extra at the 3' end (should be sufficient according to the New England
> Catalog).
> After PCR, I treat the products with Proteinase K to remove Taq polymerase
> sticking to the ends, and digest the product with the restriction enzymes
> and ligate it into a vector digested with KpnI and BglII.
> Unfortunately, the transformed ligation product doesn't yield any colonies.
> Can someone help me solve this problem?
>
> Thanks very much in advance,
>
>
> Marcel Kuiper.

It is always difficult to cut close to the ends of a PCR product.

Try:
- Kinase the ends (Oligos for PCR don't have 5' phosphates)
- Blunt end your PCR products with Klenow
- Blunt ligate into a blunt cutted/cipped vector
- Transform, miniprep, digest with KpnI and BglII (this time they cut fine)
- Low melt purify your fragment
- Ligate into the final vector between KpnI & BglII sites.

It is longer but it works...

Laurent FALQUET and Eduardo GONZALEZ (U.K. Laemmli group)


----- End Included Message -----


alternatively, try pCR 1000 from Invitrogen. It contains a
blue/white pre-cut vector containing appropriate T overhangs
to allow for direct cloning of PCR product (usually having a 3' A
overhang).

Never used it myself, but hear good things about it from
Iain McGill (I.mcgill@uk.ac.crc).

It does cost a bit though.

Mike Poidinger
Dept of Microbiology
University or Reading
From: U16788@uicvm.uic.edu
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Dissolution of DNA
Date: 14 Apr 92 18:17:57 GMT
Distribution: bionet
Organization: University of Illinois at Chicago

I had the same experience when I first tried to extract mouse
genomic DNA. I found the key is avoiding prolonged contact
with ethnol. As soon as the DNA is on the glass rod,I quickly
washed it with plenty of TE to get rid of ethnal instead of let it dry.
Then dissolve it in TE. In this way, the DNA is much easier
to be dissolved.

Zunde Wang
University of Illinois at Chicago
Newsgroups: bionet.molbio.methds-reagnts
From: adavison@selkirk.sfu.ca (Allan Davison)
Subject: Re: Microtubule depolymerisation
Organization: Simon Fraser University, Burnaby, B.C., Canada
Distribution: bionet
Date: Mon, 6 Apr 1992 23:45:36 GMT

kristoff@genbank.bio.net (David Kristofferson) writes:

>DRM21@MB1.BIO.CAM.AC.UK (David Micklem) writes:

>>Dear Net,
>>	I am making protein extracts from fruit fly ovaries and need to
>>cause depolymerisation of the microtubules.  Does anyone know whether this
>>is possible to do by changing the buffer conditions, eg by including GDP or
>>metal ion chelators?  Id like to avoid using particularly stringent
>>conditions like high salt/detergent levels if possible.
>>Many thanks for your help.

>It's been a while since I worked in this field, but fairly "low"
>levels of Ca++ (i.e., uM or less) should do the trick (I hope that
<deletion>
>memory has not failed me yet.  Adding GDP would help, but I'd bet that
>smaller amounts of Ca++ would be more effective.  Since you are making
<more good stuff deleted>

I used to prepare microtubules from sheep brains, and the problem
wasn't so much to get them depolymerized as to get them to polymerize.
My recollection of the calcium effect was as David K describes.
Moreover, polymerization required addition of GTP, Mg, (I think) and
warming to 37oC.  Cooling to 0oC depolymerized. This is standard stuff
in the literature for microtubule preparation, so check that I didn't
get it backward. Else write me direct, & I can look it up. The bottom
line is that after a short period of benign neglect, most MT are gone,
due to hydrolysis of GTP in the homogenate.
From: linder@urz.unibas.ch
Newsgroups: bionet.molbio.methds-reagnts
Subject: S. pombe library
Date: 11 Apr 92 12:00:54 MET
Organization: University of Basel, Switzerland

Dear netters,

I would greatly appreciate if somebody could indicate me sources 
where to obtain genomic S. pombe libraries.

thanks

P. Linder
Biozentrum
Klingelbergstrasse 70
4056 BASEL
Switzerland

linder%urz.unibas.ch@cernvax.bitnet
Newsgroups: bionet.molbio.methds-reagnts
Subject: RNase free DNase.
From: srckjel@chv.dsir.govt.nz
Date: 9 Apr 92 14:42:16 +1200
Organization: DSIR, Lincoln, New Zealand

We are aiming to quantify RNA in an RNA prep. but having difficulty with
co-isolated DNA.We want to use RNase free DNase to remove thisand would
appreciate comments and advice on which companies provide the best product
which is really (!) free of RNase.Thanks in advance.Jane Lancaster and Sonya
Clark.
From: parkm@GVSU.EDU (Marilyn Park)
Newsgroups: bionet.molbio.methds-reagnts
Subject: wanted sterile transfer hood
Date: 9 Apr 92 18:46:29 GMT
Distribution: bionet


          I need to purchase a sterile transfer hood (fungal cultures)
          and would appreciate advice (manufacturer, $,
          specifications)
From: MIBJOEL@UKCC.UKY.EDU (Joel Guthridge)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Breaking strepavidin/biotin bond.
Date: 12 Apr 92 17:59:18 GMT
Distribution: bionet

Tom Schneider recently asked how one could break the strepavidin/biotin
interaction to recover the biotin conjugated material.  If I remember
correctly, one can use iminobiotin instead of biotin.  The advantage
is that the iminobiotin/strepavidin interaction has a much lower affinity
than the biotin/strepavidin interaction...  allowing less harsh elution
methods to be employed.  I think Sigma and Pierce (as well as others I'm
sure) sell NHS- or hydrazine based iminobiotin for conjugate preparation.

Joel Guthridge
Univ. of Ky
Dept. of Micro. and Immuno.
Newsgroups: bionet.molbio.methds-reagnts
From: donnel@helix.nih.gov (Donald A. Lehn)
Subject: Re: how to add biotin to an oligo?
Organization: National Institutes of Health, Bethesda
Date: Thu, 16 Apr 1992 13:53:01 GMT

In article <92107.092210NETADM@DBNMEB1.BITNET> Peter Hakenberg <NETADM@DBNMEB1.BITNET> writes:
/>Does anyone have experience in adding biotin to a primer using the
/>ABI 392 Synthesizer. We want to use them for Sequencing with Dyna-beads
/>thx in advance
/>bye pi (Peter Hakenberg)


I do this all the time.  I simply use the Biotin-ON Phosphoramadite from
Clontech.  All you do is add acetonitrile and put the bottle on port
5 and specify the base as '5' in the sequence.  It seams to incorporate as
well as any other phosphoramadite.

Don.

PS. If you need to make ds biotinylated oligos, I've discovered a neat
way.  Synthesize the fist oligo with '5-ccgaattcgg-Biotin ON-n-3' at the
3' end. (n is the base on the support.  You can then make ds DNA by
annealing EcoRI linkers (BRL) and extending with polymerase.  You will
then have a biotinylated piece of DNA that you can use for affinity
purification and, since it has an EcoRI site, can be cleaved off from
the streptavidin agarose.  (Something that someone just asked how to do.)

--
Donald A. Lehn, Ph.D.                      Phone: (301) 496-2885
Bldg.37 Rm 3D20                            FAX:   (301) 496-8419
National Cancer Institute / NIH            Email: donnel@helix.nih.gov
Bethesda MD 20892
Newsgroups: bionet.molbio.methds-reagnts
From: nunn@u.washington.edu (David Nunn)
Subject: Computerized gel documentation
Organization: University of Washington
Date: Fri, 10 Apr 1992 22:04:52 GMT

  I am interested in getting feedback from labs that use systems like Eagle-Eye
(Stratagene), Imager (Appligene), Lynx (Applie Imaging), or others systems,
 for gel documentation of DNA and protein gels.  In particular; what solutions
 to datastorage, cataloging and retrieval have you come up with; cost effectiveness
 vs.plain old polaroid photos; uses for these systems that make them, in your
 opinion, worth the money; any other feelings, strong or otherwise.
From: skspoidn@susssys1.rdg.ac.uk (Mike Poidinger)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Dissolution of DNA
Date: 13 Apr 92 08:57:15 GMT
Distribution: bionet

Help.

I am trying to redissolve DNA which has been glass-rod-extracted
according to protocol 3 from Sambrook/Maniatis, and the %$#@ material
wont dissolve, wont even come off the glass rod. I am trying to prepare
medium sized genomic DNA from cell monolayers for southern Hybridization.

Any ideas on
a) Getting the DNA off the glass or
b) A better procedure for DNA preparation

would be greatly appreciated

Thanks in advance,

Mike Poidinger
Dept of Microbiology
University of Reading
From: sofer@mbcl.rutgers.edu
Newsgroups: bionet.molbio.methds-reagnts
Subject: Expression without fusion
Date: 15 Apr 92 14:06:46 GMT

Does anyone know of a lambda phage expression
vector that does -not- need require that a fusion
protein in order for one to detect recombinants.be made 

I'm looking for an insertion vector rather than
a replacement vector.

Bill Sofer
sofer@mbcl.rutgers.edu
From: herzer@urz.unibas.ch
Newsgroups: bionet.molbio.methds-reagnts
Subject: sacB vectors
Date: 15 Apr 92 11:01:59 GMT
Organization: University of Basel, Switzerland


     I had been looking for a good method of screening for double recombinants
in wild strains of E. coli and received many replies which suggested using
the sacB gene of B. subtilis which kills E. coli upon induction with 5%
sucrose.  I removed the 2.6-kb BamHI-PstI fragment from pUCD800 (Gay et al.,
1985) which includes the sacR and sacB genes, but am having some problems with
it.  Does anyone out there know of any other sacB-containing plasmids which
would be useful for cloning (possibly also temperature-sensitive).  One person
had suggested someone but I lost the name and it is no longer in the newsgroup.

Thanks for any help in this matter,
                                      Pete  "Herzer@urz.unibas.ch"
From: jnsmar14@uctvax.uct.ac.za
Newsgroups: bionet.molbio.methds-reagnts
Subject: Mitochondrial isolation using Nagarse
Date: 8 Apr 92 17:41:17 GMT
Organization: University of Cape Town

Does anyone have experience with using the proteinase Nagarse in the isolation
of mitochondria from muscle. I am trying to isolate tightly coupled, intact
mitochondria from dragonfly flight muscle, and will start experimenting 
with this, just as soon as the enzyme arrives from Sigma.
Is anyone interested in the metabolism of flight in insects? I am also working
on the hormonal regulation of the release of fuels to power flight in 
dragonflies, apart from the usual testing of maximal activities of key enzymes.
I would be most interested to contact anyone who has some experience, or is
working in this field at the moment.

Pierre Janssens
Insect Neuropeptide Research Group
Zoology Department
University of Cape Town
South Africa
From: mcgraw%gandal.dnet@SERVER.UGA.EDU
Newsgroups: bionet.molbio.methds-reagnts
Subject: RE: RT-Inverse PCR
Date: 16 Apr 92 21:51:29 GMT
Article-I.D.: server.9204162151.AA15557
Distribution: bionet


circularization is not needed.
just put your primers in with the 1st strand rxn product and amplify.

				Al
From: suter@VAX.MPIZ-KOELN.MPG.DBP.DE
Newsgroups: bionet.molbio.methds-reagnts
Subject: subject: tobacco library
Date: 16 Apr 92 10:43:27 GMT
Article-I.D.: genbank.9204161023.AA18653
Distribution: bionet



dear netters,


do any of you know of a company that sells a tobacco cDNA library ?

thanx, clemens
From: browns@ccu.umanitoba.ca (Stuart Brown)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Thermal cycler sequecing...
Date: 8 Apr 92 21:06:15 GMT
Distribution: bionet
Organization: University of Manitoba, Winnipeg, Manitoba, Canada

In article <9203312113.AA25586@umailsrv0.UMD.EDU> William_D_WARREN@UMAIL.UMD.EDU (ww40) writes:
>Does anyone out there get good results from thermal cycler sequencing ?
>
>We have been trying to sequence gel isolated PCR products using
>a gel purified end labelled primer (one of the PCR primers) and
>can't get clean sequence. We've tried 3 step and two step cycling
>with various time regimes and temperatures and still no luck.
>
>Has anyone had better luck using this technique ???

I have tried cycle sequencing using the BRL kit which employs an end labelled
primer (kinase reaction, no cleanup steps) with moderate success.

I am now trying out the Vent Polymerase Kit from NEB with high hopes (IMHO these
guys don't sell something untill all the bugs have been worked out)

In both cases, I think a key factor is using a primer for sequencing that is 
internal to the primer used to generate the PCR fragment.  Also, the PCR 
product must be very clean (I cut bands out of a gel and then used Gene-Clean)

	Hope this helps  -Stu

-- 
Stuart M. Brown                               Postdocs rush in where wise men
U. of Manitoba, Dept. Plant Science, Canada       Fear to tread. 
browns@ccu.umanitoba.ca        
				Disclaimer?? Sure: "That Disc is Mine!"
From: skspoidn@susssys1.rdg.ac.uk (Mike Poidinger)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Dissolution of DNA
Date: 15 Apr 92 09:22:06 GMT
Distribution: bionet

To the many (10) people that replied to my enquiry
concerning genomic DNA dissolution- thanks. Most involved
variations on removal of DNA from the glass rod, and
ways of redissolving it, although there was one that involved
no glass rod, and another that involved no EtOH precipitation
at all.

Once again, thanks.

Mike Poidinger
Dept of Microbiology
University of Reading
From: francis@cs.mcgill.ca (Francis OUELLETTE)
Newsgroups: bionet.molbio.methds-reagnts
Subject: new automated sequencer
Date: 4 Apr 92 17:30:08 GMT
Article-I.D.: cs.1992Apr4.173008.12526
Organization: SOCS, McGill University, Montreal, Canada

Peter Hakenberg writes:

>Some days ago I have heard of a new sequencer from milipore.
>Also, I know that hitachi has build one.
>The only thing I know is that the Milipore-Sequencer has 80 lanes (4 lane per
>probe) and the software runs on a Sparcstation. MY knowledge about the hitachi
>sequencer is zero.
>Can anyone tell me more?

Millipore does indeed have a new automatic sequencer, the
"BaseStation".  And it does work from a Sparc (Sun Microsystem).
It uses the same fluorescent chemistry as the Pharmacia ALF (ie 4
lanes per sample) but that is about where the similarity stops.
The 80 lanes (standard fluorescently labelled sanger sequenced DNA)
are loaded and they are "illuminated" by a white light, the DNA
fluoresces and a CCD camera scans the bottom of the gel and takes
"pictures" of the gel.  The "pictures" are then reconstructed to form
an "autorad" like picture which is then analyzed by their software,
which has been on the market (as a stand alone autorad gel reader) for
a couple of years. This is quite different from the ABI and Pharmacia
machines, which are taking a linear reading across the lanes.  Here
the BaseStation looks at the whole band, and makes its call from that.
The softwares work under Sun's OpenWindow.  

The product is new (released last December) and looks promissing.

I do not own a BaseStation (but if they want to lend me one I will
take it ;-) , and I do _not_ work in any way shape or
form for Millipore or Sun.  I do have their autorad reading software
(and a Sparc2) which I am just beginning to use, so I will not 
(at this time ;-) comment on it ...  All the information I have is
from talking to Millipore people and from their brochure.

hope this helps, 

regards

francis


--
| B.F. Francis Ouellette
| Manager, Yeast Chromosome I Project
| Dept of Biology, McGill University, Montreal, Qc, Canada
| francis@binkley.cs.mcgill.ca  
Newsgroups: bionet.molbio.methds-reagnts