HDTenEightyP/NetNews huggingface dataset:
From: Bo.Servenius@wblab.lu.se (Bo Servenius)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Telephone for Cruachem
Date: 27 Jan 93 13:43:50 GMT
Organization: University of Lund, Sweden
X-Xxmessage-Id: <A78C51084501380B@macservenius.wblab.lu.se>
X-Xxdate: Wed, 27 Jan 93 20:47:36 GMT
X-Useragent: Nuntius v1.1.1d13

Dear Netters:

I am trying to get hold of the telephonenumber and faxnr for
the company Cruachem - they are selling chemicals for
oligosynthesis. Please not the 800 nr as we can not use it
from abroad.

Many thanks in advance

BOSSE
Newsgroups: bionet.molbio.methds-reagnts
From: hwang@mace.cc.purdue.edu (I-shiou Ng)
Subject: Help-Making Lamda Pakaging Extract
Organization: Purdue University
Date: Fri, 22 Jan 1993 18:47:52 GMT

Hi.
Becuase the need for my reserch and lack of funding, I intend to get lamda pakaging extract by my self.  Can anyone enlight me which strain lysogenic E coli I should use and how to get extract for high efficiency pakaging?

So far I have checked Maniatis and other cook book and find following strains:
NS 428 --containing an A amber lysogen
NS 433 --containing a E amber lysogen
BHB2690
BHB2688
SMR10  

Where I can find them? Or someone can send me some strains.

Thanks

I-shiou Hwang
Entomology Department
Purdue U.
West Lafayette, IN 47907
From: BOTSALT@VM.UOGUELPH.CA (david salt)
Newsgroups: bionet.molbio.methds-reagnts
Subject: breaking small volumns of S. pombe cells
Date: 22 Jan 93 13:21:51 GMT
Distribution: bionet

I need to break small volumns of S. pombe (fision yeast), approx 1 ml, in
the presence of 100mM HCl to extract the acid soluble thiols.
I have tried grinding with no luck. Any other ideas.

David Salt, University of Guelph
Guelph, Ontario, Canada
From: mm6y+@andrew.cmu.edu (Morris F. Manolson)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: breaking small volumns of S. pombe cells
Date: 25 Jan 93 18:56:20 GMT
Distribution: bionet
Organization: Sponsored account, Biological Sciences, Carnegie Mellon, Pittsburgh, PA

> 
>%%%%%%%%%%%%%  David Writes%%%%%%%%%%%%%%
>I need to break small volumns of S. pombe (fision yeast), approx 1 ml, in
>the presence of 100mM HCl to extract the acid soluble thiols.
>I have tried grinding with no luck. Any other ideas.
> 
>David Salt, University of Guelph
>Guelph, Ontario, Canada
>%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
> 
>Hi David!
>    You should be able to use acid washed glass beads 
>0.5 mm diameter (sigma # G-9268) and vortex.
>See article by Moreno et al (1991) in Method in Enzymology
>vol 194,pg795.Ed by Guthrie and Fink (Guide to Yeast Genetics
>and Molecular Biology)  A reference to DNA/RNA/ Protein preps
>is on page 817-819.
>Good Luck
>________________________________________
>Dan Gietz
>Department of Human Genetics
>University of Manitoba
>770 Bannatyne Ave, Rm 250
>Winnipeg, Man, Canada
>R3E 0W3
>GIETZ@BLDGHSC.LAN1.UMANITOBA.CA
>_________________________________________



I purchased the mini-bead-beater from Biospecs products (918)-336-3363
for about $300.  It very effectively and quickly breaks open stationary
S. cerevisiae cells in volumes of less than one ml using 0.5 mm glass
beads.  If protein degradation is a problem, the bead-beater is alot
faster than vortexing.  I can easily get whole cell extracts of
10 to 20 mg/ml protein.  Morrie
From: g14p@alf.zfn.uni-bremen.de (Frank Prueser)
Newsgroups: bionet.molbio.methds-reagnts
Subject: insect-mtDNA PCR-Primer
Date: 26 Jan 93 08:40:19 GMT
Distribution: bionet
Original-To: methods@uk.ac.daresbury



Hi,
I'm looking for PCR-Primer fitting on proteincoding-genes 
of the insect-mt DNA. Is there anybody out there who could
send me some informations ? 
     Thanks Frank
Frank Prser
Universitt Bremen
Germany
From: jasons@bmc.uu.se (Jason E Stewart)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: ABI sequencer: reaction clean-up ??
Date: 22 Jan 93 22:23:27 GMT
Followup-To: bionet.molbio.methds-reagnts
Organization: Dept of Med Gen
NNTP-Posting-Host: mac3.medgen.uu.se

In article <1993Jan22.122927.16338@news.unige.ch>, mike@medsun.unige.ch
(michel morris) wrote:
> 
> 	We are about to do our first sequencing on an ABI 
> sequencer, using their DyeDeoxy/Taq pol. protocol. 
> 	ABI recommend (insist!) on cleaning up the reactions 
> before loading, preferably with their own spun columns - surprise, 
> surprise.
> 	Is this really necessary? How do the other protocols
> they mention, such as phenol/CHCl3 or isopropanol ppt. compare?
> What is the quickest way to get the reactions onto the machine 
> (while still getting sequence off...)?
> 
> tia,
> Mike Morris

Absolutely critical to get rid of the free nucleotides. Although we have
only used it a few times I would recommend the double phenol extraction and
warn against the iso-prop. precip. I would also recommend climbing on your
suppliers heiny and get him to install the T7 terminator kit as soon as it
comes out. With the fluorescein terminators, no cleanup is necessary.

Good luck,

Jason

----------------------------------------------------------------
Jason Stewart    	                      Father at large
Department of Medical Genetics
jasons@bmc.uu.se
From: A8101DAL@vm.univie.ac.at (Martin Hetzer)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Photography
Date: 28 Jan 93 14:14:10 GMT
Organization: University of Vienna

Usually people use the POLAROID system to take pictures of their
EB stained gels. Who has tried reflex camera for this purpose and
which film type and sensitivity is recommended?
Thanks in advance
Martin
From: GIETZ@bldghsc.lan1.umanitoba.ca
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: breaking small volumes of S.pombe
Date: 22 Jan 93 22:46:00 GMT
Distribution: bionet

%%%%%%%%%%%%%  David Writes%%%%%%%%%%%%%%
I need to break small volumns of S. pombe (fision yeast), approx 1 ml, in
the presence of 100mM HCl to extract the acid soluble thiols.
I have tried grinding with no luck. Any other ideas.

David Salt, University of Guelph
Guelph, Ontario, Canada
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%

Hi David!
    You should be able to use acid washed glass beads 
0.5 mm diameter (sigma # G-9268) and vortex.
See article by Moreno et al (1991) in Method in Enzymology
vol 194,pg795.Ed by Guthrie and Fink (Guide to Yeast Genetics
and Molecular Biology)  A reference to DNA/RNA/ Protein preps
is on page 817-819.
Good Luck
________________________________________
Dan Gietz
Department of Human Genetics
University of Manitoba
770 Bannatyne Ave, Rm 250
Winnipeg, Man, Canada
R3E 0W3
GIETZ@BLDGHSC.LAN1.UMANITOBA.CA
_________________________________________
From: mspeek@ebc.ee (Mart Speek)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: insert-positive blue cols
Date: 28 Jan 93 13:17:46 GMT
X-Newsreader: TIN [version 1.1 PL8]Lines: 43

Fiberman (wsun@jeeves.ucsd.edu) wrote:

: I have had the same experience.  I tried calling Stratagene,
: asking them where does the alpha complemetation peptide start in
: the bluescript vector and how long is the peptide, but the
: technician did not know anything.  If the peptide is short
: enough, cloning an insert into a site that's at the 3' end of
: the polylinker will not give you any white colonies.  Any one
: out there knows how long is the alpha comlementation peptide?

: -fm

    Dear Dr Fiberman;

I don't quite understand your question. But anyway, all you need
to know is that inserts in beta gal expression vectors are cloned
into mcs located after the first 5 amino acid coding sequence of
lacZ gene (e.g. see NEB catalog 1992, pp 147-148). Any disruption
of the lacZ gene if inserts are cloned into mcs of Bluescript (or any
other similar vector) results, in theory, blue to white phenotype
change. This is a basic principle (for other details see excellent
responces here by Caroline Breitenberger and Martin Kennedy)

Unfortunately however, this rule doesn't apply always. 
Quite frequently (much more frequently than one would expect) inserts
can have a ribosome "landing bad" and an in-frame initiator codon
in their 3' terminal portion which provide alternative initiation
of translation (internal initiation or promiscous initiation common
to bacterial ribosomes) of the beta gal peptide. As you may understand
I'm not talking about 3N rule and/or extra domain of beta gal (refered
above). Some folks are waisting time by picking up and analyzing
white cols/plaques which are insert negative. Thus, I wish to 
ALERT those workers:  DON'T FORGET COLONY/PLAQUE HYBRIDIZATION
when analyzing beta gal expression constructs (hybridization is
pretty much foolproof).

I must confess that at least in part, this was the reason why I raised
the question of blue/white colony screening and isolation.

MANY THANKS for EVERYONE who participated in this forum!!!!!!!!!!!!!!!

Mart (mspeek@ebc.ee                                    ESTONIA) 
Newsgroups: bionet.molbio.methds-reagnts
From: jgill@kean.ucs.mun.ca
Subject: problems with mrna quantitation
Organization: Memorial University. St.John's Nfld, Canada
Date: Mon, 25 Jan 1993 15:24:13 GMT


Fellow netters:

I have recently analysed my results during a quantitative rna analysis.  
During my analysis I used rRNA as a standard for the year to compare my 
summer hormone mrna levels.  I am dealing with temperate fish so I was 
expecting the summer hormone rna's : rRNA levels to be high during periods of 
rapid growth and the opposite to be the case during the winter months.  

However, I found just the opposite to take place.  That is to say that the 
ratios were high during the winter and low during the summer.  This is sort 
of an indication to me that in, winter flounder, the fish which I am 
presently studying rRNA varies considerably over the year.  Would anyone 
know of another mRNA which remains constant throughout the year which I 
would be able to use as a standard.

Also, I was wondering if anyone would know if it would be a good idea to 
add a foreign rna to the G.T.C. extraction mixture to show quantitative rna 
extraction when using different probes.

thanx Juan

E-mail  Jgill@kean.mun.ucs.ca
From: bio1@navi.up.ac.za (Fourie Joubert)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Cyclic Sequencing
Date: 28 Jan 93 13:37:50 GMT
Organization: University of Pretoria
NNTP-Posting-Host: bio2.up.ac.za

We are interested in buying Stratagene's Cyclic sequencing kit. We would be 
interested in any opinions about the pros and cons of using this kit, 
compared to other kits.

Fourie Joubert
Department of Biochemistry
University of Pretoria
bio1@navi.up.ac.za
Newsgroups: bionet.molbio.methds-reagnts
From: JANIEMI@FINABO.ABO.FI (Jarmo Niemi TUY)
Subject: Re: insert-positive blue cols
Organization: Abo Akademi University, Finland
Date: Thu, 21 Jan 1993 10:48:58 GMT
X-News-Reader: VMS NEWS 1.24

In <1993Jan21.090729.79@ebc.ee> mspeek@ebc.ee writes:

> 
> Hi Netters;
> 
> I would like to know whether anyone has encountered problems
> in traditional blue/white colony or plaque screening procedures
> (beta galactosidase assay) when sorting out (non)recombinant
> clones. My question is :-( Could relatively long inserts (>0.5kb)
> sometimes give rise blue (beta gal+) clones? How is it possible?
> Any theoretical or practical solutions (incl refs) to this 
> particular problem are welcome.
> 
> M. Speek
> 
> mspeek@ebc.ee (ESTONIA)
> 
Yes. A while ago I turned around a 0.6 kb insert in M13 (detaching it
from mp19 and ligating it with similarly cut mp18). When I plated both
clones on Xgal-IPTG plates the turned clone was distinctly pale blue and
the original was not. Lucky it wasn't so blue when I plated the ligation mix,
otherwise I would not have picked it. I don't know yet, why the turned clone
is blue; when I have the sequence maybe I can answer that.

Jarmo Niemi   Biochemistry, University of Turku
              SF-20500 Turku, Finland
              ++358-21-6335758
	      FAX ++358-21-633 6388
              janiemi@finabo.abo.fi

Newsgroups: bionet.molbio.methds-reagnts
From: mike@medsun.unige.ch (michel morris)
Subject: FISH background
Organization: University of Geneva, Switzerland
Date: Mon, 25 Jan 1993 13:32:39 GMT

   We are doing fluorescent in situ hybridization on human chromosomes: 
single copy, painting, interphase, the lot. 
   Although we have no trouble (at last!) getting good signals and good
specificty, we very often have a significant "halo" of cytoplasm around
metaphases, which fluoresces slightly (with biotin-labelling, avidin
development). I suspect that this may be due to cellular biotin picking
up the label.

	Anybody have any suggestions - what is causing the background?
				       how can I get rid of it?

(playing with the hypotonic shock conditions, to my surprise, does _not_ 
get rid of the cytoplasm).

t.i.a.
Mike Morris
From: GIETZ@bldghsc.lan1.umanitoba.ca
Newsgroups: bionet.molbio.methds-reagnts
Subject: RE:Looking for suitable bacterial strain
Date: 21 Jan 93 20:49:00 GMT
Distribution: bionet


%%%%%%%   Mike writes    %%%%%%%%%%%%%%

        Hello all.  I have been using Stratagene's SURE bacteria and XL-1
Blue as well as BRL's DH10B electrocompetent cells.  What I need to use is
a strain that tolerates plasmids with minimal rearrangement (like SURE),
which works well with electroporation and (the kicker here...) which
does not have dam or dcm methylation.  If anyone has used a strain for
electroporation which is dam- and dcm-, I'd appreciate hearing from you.

Mike Coady
COADY@ERE.UMONTREAL.CA
%%%%%%%%%%%%%%%%%%%%%%%%%%%%
Hi Mike
        I have used JM110, which is dam,dcm , in a quick and dirty
electroporation and it worked fairly well.
  Unfortunately, I don't have any numbers for you.
I just scraped off a big blob of cells from a fresh O/N plate and
washed with 1 ml of cold water twice and resuspended in 50 ul
of 10% glycerol and EPed.  I plated 2 ul of the 1050 ul of SOC culture
and got a fair number of amp r colonies, if I remember correctly.  
Unfortunately
It's not as good as DH5 alpha done this way.
The genotype is F' traD36 lacIQ|(lacZ)M15 proab/rpsL (Str r)
    thr leu thi lacY galK galT ara fhuA tsx dam dcm supE44
    |(lac-proAB).

Dan Gietz
U of Manitoba
GIETZ@BLDGHSC.LAN1.UMANITOBA.CA
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: PCR sequencing
From: chai_z@wehi.edu.au
Date: 19 Jan 93 11:20:13 +1000
Reply-To: in%"dsct@dbl.cc.rochester.edu"
Organization: Walter & Eliza Hall Institute

In article <1993Jan16.183926.14051@galileo.cc.rochester.edu>, DSCT@db1.cc.rochester.edu (DAVID SCOTT) writes:
> I would like to know if anyone has any experience going from cDNA to sequence.
> I am currently (the gel is running now) doing the following.
> 1. Generated cDNA with specific 3'end of gene primer.
> 2. 20 rounds of PCR with both primers
> 3. purification of PCR product. (geneclean)

Normally I do not purify the DNA if I got only one band. It is critical to get
rid of the free primers present in your PCR reaction. You may use Promega 
Saphacryl (S200 or S300 or S400) spun column, or Magic PCR Preps, or other
Primer Erase columns to separate your DNA products from the small molecules
including the primers (very quick and convinient).


> 4. addition of both primers ( the 5' of which has been end labled with 32P )

For sequencing reaction, ONLY ONE primer is needed in a reaction. You may set 
two sets of reaction using one of the two primers for each reaction so that you
 may get sequences from both ends.


> 5. PCR in 4 tubes with the different dNTP's and ddNTP's(diffent ddNTP in 
>              each tube). PCR done for 20 cycles. 

You may have noticed that the proportion of each ddNTP in the reaction is not
exactly the same. I normally did PCR sequencing for 30-40 cycles, depending on
the amount of your template DNA and the efficiency of primer binding
(temperature, etc)


> 6. I then loaded 3microliters of each reaction onto the sequenceing gel (8%)
> 7. I don't know why it won't work, but I have a feeling I am missing something.

Have you gotten a smear or what?
> 
> Any help/experience with doing this would be apreciated.
> 	
> 					Gavin Fischer
> 					University of Rochester Cancer Center
> either post or e-mail to dsct@db1.cc.rochester.edu
> 		         dsct@uordbv 
> 
> 	Thank you


I use New England Lab's CircumVent kit for sequencing reactions. I found the
ratio of DNA and Primer (~300ng/2pmoles) was important for a clear reading. The
 best I've gotten is over 400 bases clear reading (two runs of electrophoresis).

I hope this helps.

Zhonglin Chai
Melbourne
From: vmiao@oregon.uoregon.edu (Vivian Miao)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: CH3-ation of cytosine residues...how?
Followup-To: bionet.molbio.methds-reagnts
Date: 22 Jan 1993 04:38:38 GMT
Organization: Institute of Molecular Biology
Distribution: world
NNTP-Posting-Host: fp1-molbio-30.uoregon.edu

In article <1j7ju0$73b@agate.berkeley.edu>, nigel@pathos (niggley mon)
wrote:

> Is there an easy way to methylate all the cytosine residues (5-m) of
> plasmid DNA in vitro?  If not, can I get 'coli to do it for me?

You can make 5 meC DNA by PCR (substitute 5meC in the 
nucleotide mix for C).  NEB also sells a CpG methylase that may suit your
purpose, if all the positions you are interested in are CpG dinucleotides.
From: ser3@CUNIXF.CC.COLUMBIA.EDU (Stuart E Ravnik)
Newsgroups: bionet.molbio.methds-reagnts
Subject: (none)
Date: 28 Jan 93 19:41:09 GMT
Distribution: bionet


unsubscribe
From: jxc15@po.CWRU.Edu (Jens Cavallius)
Newsgroups: bionet.molbio.methds-reagnts
Subject: recA, dut-, ung- E. coli strain
Date: 26 Jan 93 20:48:30 GMT
Organization: Case Western Reserve University, Cleveland, Ohio (USA)
NNTP-Posting-Host: slc12.ins.cwru.edu


Hello all,
I have been using CJ236 cells but having problems with recombinations
resulting in loss of insert.  What I would need is a strain like the
CJ236 but that is also rec-.
If anyone has a strain like that or know of one or where to bye one,
I'd appreciate hearing from you.

Jens Cavallius, Ph.D.
CWRU
jxc15@po.cwru.edu
or
cavallius@cwbio.bioc.cwru.edu
Tel: (216) 368-3466		Fax: (216) 368-4544
--
From: lapointe@THORIN.UTHSCSA.EDU ("David Lapointe")
Newsgroups: bionet.molbio.methds-reagnts
Subject: Needed - Human Tumor Cell Line Database
Date: 26 Jan 93 20:25:13 GMT
Distribution: bionet

I have received two calls today from researchers
interested in a datbase of tumor cell lines.

Aside from the ATCC catalog is there a database of tumor 
cell lines?


 .david
+-------------------------------------------------------------------+
| David S Lapointe PhD                     lapointe@uthscsa.edu     |
| Computing Resources                      lapointe@uthscsa (BitNet)|
| 7703 Floyd Curl Drive                                             |
| UTHSCSA                                  (512) 567-2200 (voice)   |
| San Antonio,TX 78284-7832                (512) 567-2204 (fax)     |
+-----*** The network to hell is wired with good intentions ***-----+

Newsgroups: bionet.molbio.methds-reagnts
From: cbreiten@magnus.acs.ohio-state.edu (Caroline A Breitenberger)
Subject: Re: insert-positive blue cols
Organization: The Ohio State University
Date: Thu, 21 Jan 1993 15:24:04 GMT

My technician made an intersting observation in this regard.  He was
preparing a construct for a yeast knock-out like this:
    appr. 500 bp frag - URA3 - appr. 500 bp frag - pBS
He had a URA3 clone in pBS, used PCR to get the upstream gene
fragment and cloned it in, which worked OK.  Then, he used PCR
to get the downstream fragment, tried to put it in the doubly
digested clone.  No luck!  We sat down and went over the whole
procedure, and the strategy he had used made sense, but he kept
talking about screening *white* colonies.  He had obtained blue
colonies in the last step, but since he had not expected any, he
assumed they were contaminants (this from a guy who never had
problems with contamination).  Sure enough, when he looked at blue
colonies, they all had the desired insert.  (The white colonies he
screened were either vector that had only been cut with one enzyme
or aberrant religation of "incompatible"ends.)
In this case, the total insert was about 2 kb.  I did not check the
sequence, but I assume that putting in the downstream fragment
introduced a Shine/Dalgarno and AUG in the right reading frame to
read into the beta-gal gene.  (If you look at the sequences of
many of these lacZ cloning vectors, it seems pretty clear that the
sequence upstream of the cloning site is not necessary for beta-gal
function.)

In another case, a graduate student who is using the Clontech TA cloning
kit (:-)) noted that clones containing his desired PCR-derived insert
(appr. 250 bp) exhibit a pale blue phenotype.  In this case, I think
that the insert provides a continuous open reading frame which results
in expression of the alpha-complementation peptide with an N-terminal
extension.  The pale blue color suggests that this peptide is less
efficient than that of the parental vector.  (The opposite orientation
of the insert presumably gives white colonies - I forgot to ask him.)

Caroline Breitenberger, Department of Biochemistry
Ohio State University, 484 W. 12th Ave., Columbus, OH 43210, USA
614-292-9473.......caroline+@osu.edu
From: GIETZ@bldghsc.lan1.umanitoba.ca
Newsgroups: bionet.molbio.methds-reagnts
Subject: RE: Long Seq Gels
Date: 25 Jan 93 19:09:00 GMT
Distribution: bionet

------------------------------------------------------------------------------
I'm running fairly long (52 cm) sequencing gels with labelled DNA
fragments.  Right now we have to cut the top 10 cm because we don't have
X-ray film that's long enough.  I recall seeing X-ray film in rolls
somewhere.  Does anybody recall brand, size and price?

Also, we don't have a gel dryer that accomodates long gels.  Any
alternative drying method?

Cheers everybody

Pablo A. Scolnik
scolnipa@esva.dnet.dupont.com
_____________________________________________________

Pablo, 
    Why don't you try cutting the gel with a razor blade
and putting your gel to film in pieces.  If you load a sample
twice (at two different times) the place of the cut should
be no problem ( Overlap with the other load).  This worked 
for me when I was using King Kong gels.

________________________________________
Dan Gietz
Department of Human Genetics
University of Manitoba
770 Bannatyne Ave, Rm 250
Winnipeg, Man, Canada
R3E 0W3
GIETZ@BLDGHSC.LAN1.UMANITOBA.CA
_________________________________________
Newsgroups: bionet.molbio.methds-reagnts
Subject: insert-positive blue cols
From: mspeek@ebc.ee (Mart Speek)
Date: 21 Jan 93 09:07:27 +0200
Keywords: blue/white selection 
Summary: beta gal assay
X-Newsreader: TIN [version 1.1 PL8]Lines: 15


Hi Netters;

I would like to know whether anyone has encountered problems
in traditional blue/white colony or plaque screening procedures
(beta galactosidase assay) when sorting out (non)recombinant
clones. My question is :-( Could relatively long inserts (>0.5kb)
sometimes give rise blue (beta gal+) clones? How is it possible?
Any theoretical or practical solutions (incl refs) to this 
particular problem are welcome.

M. Speek

mspeek@ebc.ee (ESTONIA)