HDTenEightyP/NetNews huggingface dataset:
From: S0679400@deimos.ucc.umass.edu (s0679400)
Subject: help
Organization: University of Massachusetts at Amherst
Date: Sun, 16 Aug 1992 17:01:45 GMT
X-News-Reader: VMS NEWS 1.20

I have been wondering if any of the protein A and protein G alternatives bind
to chicken immunoglobulins with good affinity?  I have been trying to develop
an immunoprecipitation method using chicken serum.  The anti-chicken
immunoglobulin that I have been using seems to precipitate some other proteins
as well interfering with my results.  I would appreciate all the help sent to
me by the GURUs of immunoprecipitation method.
	Thanks a lot
	Amrut
From: APPLING@UTBC01.CM.UTEXAS.EDU
Newsgroups: bionet.molbio.methds-reagnts
Subject: yeast cDNA library
Date: 14 Aug 92 19:05:14 GMT
Distribution: bionet

We are trying to clone a gene from Saccharomyces cerevisiae by complementation
of an E. coli mutant.  We have been unable to do this using genomic libraries.
If anyone knows of a cDNA library of yeast in an E. coli expression plasmid,
we would like to hear from you.  We can be reached at:
	appling@utbc01.cm.utexas.edu
Thanks,
	Dean Appling
	Univ. of Texas
	Austin, TX 78712
	512-471-5842
From: daryl@RT2.MENZIES.SU.EDU.AU (Daryl Webb)
Newsgroups: bionet.molbio.methds-reagnts
Subject: HOS cells
Date: 21 Aug 92 15:29:33 GMT
Distribution: bionet

Sorry if this is a little off beat for this newsgroup but I'm hoping that its
wide readership might come up with something.  grovel, suck :-)*

We wish to obtain the following cell line for virological studies:

HOS (human osteogenic sarcoma) cell line described in RM McAllister et al
Cancer (1971) 27:397-402 and listed as ATCC CRL 1543

Though this cell line is available from ATCC, it would be more convenient (no
AQIS etc) to obtain it from another Australian institution.

So all you Aussie Labbies do me a favour and have a look in the bottom of
that LN2 tank in the corner eh.

To stop me wasting any more bandwidth please E_mail replies.

Ta muchly

-- 
                                                     |      |
  Daryl Webb  (daryl@menzies.su.edu.au)               \    / 
  Menzies School of Health Research                    \__/   
  P.O. Box 41096                                        ||   <<
  Casuarina N.T. 0811            ----------------------- *  <<
  Australia                                             ||   <<
  Voice : 61_89_228196                                 /||\ 
  Fax   : 61_89_275187                                * || o
                                                     /  ||  \    
Newsgroups: bionet.molbio.methds-reagnts
From: jgraham@bronze.ucs.indiana.edu (the End)
Subject: Uni vs. bidirectional Exo III deletions
Keywords: exonuclease bidirectional deletions
Organization: Indiana University
Date: Wed, 19 Aug 1992 18:39:01 GMT

Hello networkers,

I recently was rather successful in creating some deletions in an
Bluescript based plasmid using the Exonuclease III/ Mung Bean
procedure as described by Strategene in their literature that
accompanies the Bluescript vector. The problem is that I would like to
create BIdirectional deletions centered at a unique site and the
twenty or so clones I have sequenced all have deletions extending in
either one or the other direction from the target restriction site.
This site generates the appropriate ends for Exo III deletion in both
directions.

I am using 30U of exonuclease for 2.5 micrograms (1 pmol) of
linearized plasmid DNA and digesting at room temperature for intervals
under 60 seconds.

Samples taken at 2 secs show a pattern of unidirectional deletion of
between 1 and 45 bp, while longer incubations do not show any
deleteion. These deletions in samples taken at the first time point
appear to be products of a step-wize removal of residues, as many
identical clones are seen that are deletions of either 12, 30, or 45
base-pairs. I have not detected any of intermediate size in the 75
clones I have looked at.

I have tried reducing the enzyme concentration and increasing the
reaction time, and reducing the temperature to 16 C and increasing the
enzyme concentration three fold, but have yet to analyze these
reaction products.


Has anyone successfully created deletions in both directions with Exo
III or experienced similar problems ?

Thanks much,

Jim
J. E. Graham
Biology/Chemistry Departments
Indiana University
.............."Il n'y a pas des sciences apliquees, mais il y a..............
.................des applications de la science". L. Pasteur.................
From: kramer@oj.rsmas.miami.edu (Jack Kramer)
Newsgroups: bionet.molbio.methds-reagnts
Subject: polysaccharides in DNA extractions
Date: 19 Aug 92 17:39:42 GMT
Organization: University of Miami, RSMAS


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.
From: William_D_WARREN@UMAIL.UMD.EDU (ww40)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Uni vs. bidirectional Exo III deletions
Date: 19 Aug 92 23:18:00 GMT
Distribution: bionet

>Hello networkers,
>
>I recently was rather successful in creating some deletions in an
>Bluescript based plasmid using the Exonuclease III/ Mung Bean
>procedure as described by Strategene in their literature that
>accompanies the Bluescript vector. The problem is that I would like to
>create BIdirectional deletions centered at a unique site and the
[stuff deleted] 
>Has anyone successfully created deletions in both directions with Exo
>III or experienced similar problems ?
>
>Thanks much,
>
>Jim
>J. E. Graham
>Biology/Chemistry Departments
>Indiana University

Jim...
I suspect that your problem is that you are using too little
of the Exo III - because unless there is an excess of enzyme you
won't get a set of uniform deletions. For UNI-directional deletions
I typically use 100 U of Exo III per ug of DNA (typically 4-8kb). Also
the rate of digestion is generally linear with temperature, progressing at
around 300bp/min at 35oC, 500bp/min at 39oC. And although I have never
set out to make BI-directional deletions myself, I have seen some
when co-workers have forgotten to perform the second digestion to
produce a protestive 5'overhang, so I can assure you that IT DOES WORK.

Another mistake I've seen people make is to add 10xExoIII buffer
(which in my protocol contains 7mM MgCl2) to their DNA that has been
suspended in TE - this results in NO DIGESTION AT ALL since the Mg2+
gets all sucked up by the EDTA. Either resuspend the precipitated DNA
in 1xBuffer or in H2O then add the 10xBuffer.

I have had troubles when trying to generate deletions that have
alot of secondary structure. Do you know the sequence of your DNA ?

Hope this helps

Bill Warren
Center for Agricultural Biotech
University of Maryland

(ps "DNA Dipstick" gets my vote for "Most Useless Kit of the Year" too!)
From: bsh@MED.PITT.EDU (Basavareju Shankarappa)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: help
Date: 16 Aug 92 20:10:53 GMT
Distribution: bionet

> I have been wondering if any of the protein A and protein G alternatives bind
> to chicken immunoglobulins with good affinity?  I have been trying to develop
> an immunoprecipitation method using chicken serum.  The anti-chicken
> immunoglobulin that I have been using seems to precipitate some other proteins
> as well interfering with my results.  
>  Amrut
 

Have you considered other alternatives like the immobilized anti-chicken 
immunoglobulin.  As far as I knew, one company used to make this preparation
i.e., latex sphere bound anti-chicken immunoglobulin.  I think this method
may increase the specificity of your assay and thus more reliable results.

The place I used to buy these from is Kirkegaard and Perry Laboratories
It is in Gaithersburg Maryland.  Tel: (800) 638-3167.

Hope this helps
Good luck
Raj Shankarappa
University of Pittsburgh
Newsgroups: bionet.molbio.methds-reagnts
From: ca566@cleveland.Freenet.Edu (Michael Holloway)
Subject: Re: 33P
Reply-To: ca566@cleveland.Freenet.Edu (Michael Holloway)
Organization: Case Western Reserve University, Cleveland, OH (USA)
Date: Tue, 18 Aug 92 23:24:10 GMT


In a previous article, afc@gnv.ifas.ufl.edu (Andrew F. Cockburn) says:

>In article <n62m_xn@lynx.unm.edu>, bhjelle@carina.unm.edu writes:
>> In article <1992Aug10.184231.25639@gserv1.dl.ac.uk> skspoidn@uk.ac.rdg.susssys1 ("Mike Poidinger") writes:
>>>WRT comments on 35-S, sticky gels and 33-P
>>>
>>>1) I don't think they did mean 32-P. They meant 33-P. 
>>>It's new on the market, supposed to have 5 times the 
>>>strength of 35-S, which is a lot less than 32-P
>> 
>> Also costs a lot more. I nixed that one right away when
>> NEN quoted a price.
>
>I looked in my 92/93 NEN catalog and saw that 33-P gamma ATP
>is actually cheaper than 32-P.  Of course that is list price,
>and I have never in my life paid list price for radioactivity.
>Was your quote for dNTPs instead?
>
>In any case, I would expect price to drop rapidly if demand 
>picks up.

The alpha dATP 33P is more expensive and they where not giving any
discounts as of last month (at least not to SUNY@Stony Brook).  They do,
however give a free sample, which is the only reason I've been able to try
it.

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: levenson@duke.cs.duke.edu (Richard Levenson)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Which PCR machine ?
Date: 12 Aug 92 14:37:59 GMT
Distribution: usa
Organization: Duke University Computer Science Dept.; Durham, N.C.

No doubt this is a FAQ, but which PCR machine should we buy.  We have a
Peltier one already which has given us some trouble.
Has anyone had any experience with the forced-air models?
Reliability?
Ease of use?
Price?

Thanks ever so.  Replies can be posted to the net or e-mailed to
levenson@cs.duke.edu.


-- 

Yo, adrian
From: coyne@uctvax.uct.ac.za
Newsgroups: bionet.molbio.methds-reagnts
Subject: HELP: ligation (used to be Re: cip & geneclean)
Date: 13 Aug 92 10:00:10 +0200
Distribution: bionet
Organization: University of Cape Town

Dear Bruce and fellow netters,

We were very pleased to see the following posting by Bruce Roe as we are
currently experiencing similar problems to those reported by Michael; except
that we are trying to clone 10 kb gel purified, BamHI chromosomal DNA
fragments into BamHI cut and cipped Bluescript.  Although Bruce's suggestions
are extremely helpful, I have a question relating to point #3 below:

In article <10AUG199208480766@aardvark.ucs.uoknor.edu>, broe@aardvark.ucs.uoknor.edu (Bruce Roe) writes:
> In article <1992Aug9.912.1309@channel1>, "michael coyne" <michael.coyne@channel1.com> writes...
>>Hello fellow molbio.metters:
>> 
>>I have a question to which I have been unable to get a clear answer.  I
>>do not expect a cut-and-dried answer, but perhaps a consensus would be
>>helpful.
>> 
>>We have been attempting to clone a fragment of DNA into a high
>>copy-number vector (a derivative of the pUC plasmids).  We have had some
>>problems with incomplete digestion of the vector causing high
>                ^^^^^^^^^^^^^^^^^^^^
>>background, thus making identification of recombinant clones difficult
>>(using lacZ blue/white identification).  
> 
> Suggestions:
> 	1. Try a series of simple control ligation experiments where
> 	   you linearize the vector with the restriction enzyme for
> 	   various times at various enzyme/vector rations.  Then just
> 	   do a transformation without the ligation step.  This will
> 	   help you determine the optimum digestion conditions to
> 	   minimize any un-cut vector.
> 	2. Once you've found digestion conditions where there is minimum
> 	   un-cut vector, then do a time study for BAP or CIP
> 	   de-phosphorylation.  (usually need 30min, 1hr, 2 hr)
> 	   Do a ligation/transformation for each time point without
> 	   added insert to see the efficiency of the phosphatase.
> 	3. Repeat the ligation/transformation using the de-phosphorylated
> 	   vector with minimal background produced in step #2 and this
> 	   time use varying amounts of insert for the ligation, holding
> 	   the vector amount constant.

QUESTION:	What is the recommended starting concentration of total DNA 
(or that of insert & vector)?  We have been starting with 5 pM of total DNA and
then performing 2-fold serial dilutions of the insert DNA while keeping the
concentraion of vector DNA constant.  However, we are continually plagued with
low numbers of transformants (and inserts) following transformation of E. coli
LK111 competent cells.

(rest of Bruce's original posting deleted; still available on
molbiol.meths-reagnts)

Thank you (in advance) for any assistance with our problem,
Vernon Coyne
coyne@uctvax.uct.ac.za
From: moosp@matt.ksu.ksu.edu (Philip Joseph Moos)
Newsgroups: bionet.molbio.methds-reagnts
Subject: electroblotting southerns
Date: 13 Aug 1992 12:34:23 -0500
Organization: Kansas State University
NNTP-Posting-Host: matt.ksu.ksu.edu

Hi,

I never received any responses on protocols for electroblotting southerns --
just - "I'm interested too...be sure to post summary".

Does anybody have a protocol that works well??  I can tell you the protocol
in the Hoeffer manual does not -- at least not unmodified.

Thanks in advance,

Philip Moos
moosp@matt.ksu.ksu.edu

(I will post a summary if I get any responses.)
From: bioysl@lure.latrobe.edu.au
Newsgroups: bionet.molbio.methds-reagnts
Subject: PBR322 primers wanted, URGENTLY!!!!
Date: 19 Aug 92 05:02:20 GMT
Organization: VAX Cluster, Computer Centre, La Trobe University

Hi! Friends!

	Could anyone give me some primers which are arround the BamHI site in
PBR322 Tet region?  I need them for PCR.

	Thanks in advance.

Yong Shu LIU
Biochem Dept
La Trobe Uni.
Bundoora, Vic.
Australia 3083
(613) 479 1175,    479 2467(fax)
From: kramer@news.miami.edu (Jack Kramer)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: polysaccharides in DNA extractions
Date: 22 Aug 92 00:13:38 GMT
Organization: University of Miami, RSMAS

In article <1992Aug21.154346.18547@ccu.umanitoba.ca> browns@ccu.umanitoba.ca (Stuart Brown) writes:
>
>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.
>

Thanks for all the responses.  The majority have been along these same
lines.  I have used the CTAB procedures, even increasing the CTAB to
5% and 3x the normal buffer to tissue ratio.  This helps for most
plants.  Lyophilizing the tissue and keeping it frozen under liquid
N until thawed in the hot CTAB helps even more.  However, in some
really slimey cultivars, after 2 extractions of the CTAB with
chloroform/isoamyl alcohol, the first isopropanol precipitation still
produces a majority of slime.  Some of the white DNA can be fished
out of the slime and works in subsequent PCR but most of it is
buried in the slime.

Further suggestions are solicited.
From: wmelchior@NTBTOX.NCTR.FDA.GOV ("Bill Melchior, NCTR/FDA")
Newsgroups: bionet.molbio.methds-reagnts
Subject: RE: polysaccharides in DNA extractions
Date: 19 Aug 92 17:34:51 GMT
Reply-To: wmelchior@NTDOC.NCTR.FDA.GOV
Distribution: bionet

>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?

DNA and RNA are not polysaccharides in the usual sense, since they're
linked through phosphate esters rather directly from sugar to sugar.
I don't know for sure, but would be surprised if enzymes that digested
one type of molecule would harm the other.  Trying chemical degradation 
of polysaccharides without damaging DNA could be riskier.

Glassmilk purification might work.  Hydroxyapetite adsorption would 
probably be specific for the DNA.
________________________________________________________________________________
The opinions stated are mine, not those of NCTR or its sponsoring organizations.

Bill 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

Newsgroups: bionet.molbio.methds-reagnts
From: achernar@aix00.csd.unsw.OZ.AU (Brono Gatea)
Subject: Maxam & Gilbert kit recommendation
Organization: University of New South Wales
Date: Wed, 12 Aug 1992 14:28:24 GMT

I am about to start some DNase I footprinting, and am looking for an easy way
to obtain the Maxam & Gilbert reference lanes. I have done some M & G before
and hated it! I have heard that there are kits around that make the job easier.
Anyone on the net with recommendations?             

Many thanks

Bruno Gaeta
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Automated sequencing
From: morris@medsun.unige.ch (mike morris)
Date: 20 Aug 92 15:48:35 +0200
Distribution: bionet
Organization: University of Geneva, Switzerland

In article <9208192048.AA03649@sol.UVic.CA>, jdboer@SOL.UVIC.CA (Johan deBoer) writes:
> 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

The Pharmacia machine looks very interesting for SSCPs, 'cos of its
control of gel temperature. Anyone out there tried it?

Mike M.
From: stine@jeeves.ucsd.edu (Blaine Stine)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: MacVector
Date: 14 Aug 92 16:41:07 GMT
Organization: University of California, San Diego

In article <1992Aug13.144622.19374@yang.earlham.edu> derekr@yang.earlham.edu writes:
>We've been experiencing difficulties with the program MacVector.  Does
>anyone know of a similar program for the Macintosh that aligns sequences?
>Thank you.
>
>Derek Reubish
>Dept. of Biology
>Earlham College

If you are having problems or have found a bug why don't you get
in touch with IBI? You didn't mention if you are having problems
with the Assembly Lign addition to MacVector or the original
application. I can't imagine having "difficulties" with
MacVector and just deciding to chuck the whole thing (all $3000
worth or something) and looking elsewhere!  There are quite a 
number of MacVector users here at UCSD all of which  *really*
like the application.

Blaine Stine
stine@jeeves.ucsd.edu
Newsgroups: bionet.molbio.methds-reagnts
From: ca566@cleveland.Freenet.Edu (Michael Holloway)
Subject: Baby powder those sticky gels
Reply-To: ca566@cleveland.Freenet.Edu (Michael Holloway)
Organization: Case Western Reserve University, Cleveland, OH (USA)
Date: Fri, 21 Aug 92 00:26:12 GMT


I've received an E-mail message about the "sticky sequencing gel" problem
that I was asked to pass on.  Seems like a perfectly reasonable solution.

eturn-path: <weber@calshp.cals.wisc.edu>
Received: from calshp.cals.wisc.edu by ccmail.sunysb.edu (PMDF #2330 ) id
 <01GNQRIJELF48ZFRE6@ccmail.sunysb.edu>; Tue, 18 Aug 1992 22:29:36 EDT
Received: by calshp.cals.wisc.edu; id AA27711; 16.6/42; Tue,
 18 Aug 92 21:25:05 -0500
Date: 18 Aug 1992 21:25:05 -0500
From: weber@calshp.cals.wisc.edu
Subject: sticky gels
To: mhollowa@ccmail.sunysb.edu
Message-id: <9208190225.AA27711@calshp.cals.wisc.edu>
Content-transfer-encoding: 7BIT

        Hi!  I would have posted this on the net except that I
don't know how and made a real mess once trying to post something.

Maybe if it works for you you can pass it on.
        Anyway, what we do with sequencing gels is dry them for
45 mins to an hour, peel off the Saran Wrap, and sprinkle
with baby powder (which is basically cornstarch); rub it around
until all the parts of the dried gel are covered and not sticky,
shake off the excess powder, and place directly on film.  It works
for us.
        Good luck,
        Leigh
Weber
----------------------------------------------------------------------------
-- 
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: byrne@soma.umdnj.edu (Bruce Byrne)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: 2nd round of PCR
Date: 18 Aug 92 13:22:00 GMT
Distribution: bionet

walterh@U.WASHINGTON.EDU (Walter Hill) writes:

>Dear fellow net-rats:
>A colleague of mine is amplifying rotavirus gene using RT-PCR and that works
>well and a second round of PCR cycles using nested primers also works well.
>However, when the second amplification is performed with primer identical to
>the first amplifcation, no or very little amplification occurs.
>Can anyone provide an explaination for this phenomenon?

Maybe.  If the original target were rare or not a very good fit to your 
primers, your colleague likely formed some primer dimers.  Those unwanted
PCR products are perfect targets for the second round of amplification by
the same primer pair.

Added note of caution.  Any second round of amplification adds great
complexity to handling carryover contamination.  

Cheers!

Bruce Byrne
Newsgroups: bionet.molbio.methds-reagnts
From: num208jn@MBDS.NRC.CA (John Nash)
Subject: Re: Peltier PCR Instruments - Possible explaination of mixed reviews
Reply-To: num208jn@MBDS.NRC.CA
Organization: NRC, OTTAWA, ONTARIO, CANADA.
Date: Wed, 19 Aug 92 13:59:27 GMT

In article <92232.080048BACKD@QUCDN.QueensU.CA>, <BACKD@QUCDN.QueensU.CA> writes:
>I had the first MJ machine placed in Canada. The peltier units failed
>about every 4 to 6 months. MJ was VERY accomodating in getting my unit
>repaired and returned to me ASAP at no charge. They continued to do so
>even after warrantee and even included software upgrades no charge.
>The peltier problems were so bad that the sales rep at the FASEB booth
>recognized my name and began a spontaneous round of apologies.
>
>The last peltier was installed during the summer on 1990. Since then
>we have had no further problems. I believe the MJ machine is one of
>the most versatile machines on the market. We use it for PCR, sequencing
>controlled rate annealling and occasionally for restriction digestion
>run at odd temperatures.
>
>Don Back
>Department of Biochemistry
>Queen's University
>Kingston, Ontario. CANADA.

Since we're recounting peltier stories...

I had one of the first COY TempCyclers (peltier unit) machines.  The
peltiers used to jam... at one time melting the tubes, and another
time freezing the block at -20decC.  COY/Diamed fixed the machine,
kept me happy with a loaner, and basically gutted my Model 50 and
replaced the innards with Model 60 parts.  It hasn't failed since
(touch wood), and I use it for PCRs, RAPDs, Cycle sequencing etc.

However.... I will NEVER again run a PCR/RAPD/Cycle sequence without
my temperature readout & chart-recorder hooked up!


John Nash                            | Email: Nash@biologysx.lan.nrc.ca.
National Research Council of Canada  | Mail to "num208jn@mbds.nrc.ca"
Ottawa, Ontario, Canada              |    is forwarded automatically.

Disclaimer:  All opinions are mine, not NRC's!
Confession:  I've NEVER used a UNIX machine!