HDTenEightyP/NetNews huggingface dataset:
From: bsh@MED.PITT.EDU (Basavaraju Shankarappa)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: whence Coomassie?
Date: 7 Oct 92 17:49:36 GMT
Distribution: bionet

> I have a question that follows from the Coomassie one.
> At the moment I am running protein gels on Pharmacia's PhastGel
> system. It seems that high sensitivity silver stainning works
> better if the gel is Coomassie dyed first. 
> 
> Does anyone know why this is so? The Pharmacia reps didn't have a
> clue.

I second this observation.  I had seen it with both the Phastgel and 
the regular 1.5 mm polyacrylamide gels.  
I could be wrong but the line of reasoning I consoled myself was that
since there is a directt correlation between the length of fixing
and the quality of silver staining, the staining and destaining bby
coomassie does take out a lot of junk.  So it had always been better
silver after coomassie.
Another reason is that specially for some proteins it is next to impossible
to destain the gels completely to remove the stain.  So I think the coomassie
blue reacts with the silver stain reagent and intensifies the signal.
Is there a better explanation?  I will be interested.
Raj Shankarappa
Univ of Pittsburgh.

Newsgroups: bionet.molbio.methds-reagnts
From: jgraham@bronze.ucs.indiana.edu (the End)
Subject: Tightest promoter repression ?
Organization: Indiana University
Date: Mon, 12 Oct 1992 03:09:17 GMT

I need to find a promoter which is very tigthly regulated for use in reducing
the expression of downstream Lac sequence to a very low level.

I currently am using the tac promoter with the lac IQ allele carried at pUC
copy number in a second plasmid. Even with this repressor level I am seeing a
certain amount of background lac Z expression in the absence of inducer.

Tell me your experiences in using regulated promoters. I prefer to use a 
rather strong single promoter (eg. not Pgal) that will function well
in vitro.

Thanks much,


Jim
From: kim@m44.unm.edu
Newsgroups: bionet.molbio.methds-reagnts
Subject: Differential Display PCR
Date: 8 Oct 92 18:58:02 GMT
Reply-To: kim@m44.unm.edu
Organization: UNM Cancer Center, Albuquerque, NM

Hello Group:

Has anyone tried out the "Differential Display PCR" technique of <someone> and
Pardee (SCIENCE 257:967-?)?  We are giving it a try right now, but I'd like to
see if anyone else has given it a shot.

Thanks
Daniel Kim
KIM@FLOVAX.LANL.GOV
From: ab4u+@andrew.cmu.edu (Anand K. Bachhawat)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Savant Pump
Date: 12 Oct 92 16:50:39 GMT
Organization: Biological Sciences, Carnegie Mellon, Pittsburgh, PA

Dear Netters,
We're thinking of getting a Savant oil free pump for drying gels. Does
anyone have any experience with these pumps and are they better than 
getting the regular oil pumps with a vapour trap? I understand that
there was a lot of discussion on this several months ago but I missed
it. If someone would be willing to summarize some of the salient
features that came out of those discussions that would be greatly
appreciated. Thanks a lot, Anand Bachhawat,CMU
From: s882076@yallara.cs.rmit.OZ.AU (Sean Davidson)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: hybridization problems
Date: 10 Oct 92 03:11:10 GMT
Distribution: bionet

Further to this discussion about different membranes, I recently began using
the Digoxigenin-based system for Northern blotting. Initial attempts used
Hybond N+, which is recommended for this procedure, but signal was always
virtually undetectable amongst high background. When I began using
Zetaprobe (Biorad) instead, results were (are) great.
  So do you think this may have just been the batch of Hybond, or why are
the results so much different?

Sean
Newsgroups: bionet.molbio.methds-reagnts
From: zxmkr08@mailserv.zdv.uni-tuebingen.de (Cornelius Krasel)
Subject: Re: whence Coomassie
Organization: Comp. Center (ZDV) U of Tuebingen, FRG
Distribution: bionet
Date: Sun, 11 Oct 1992 17:21:46 GMT

In <1992Oct5.115704.15875@gserv1.dl.ac.uk> BI1RGW@ibm.shef.ac.uk writes:

>2. How good is the linearity for conc:stain? Presumably this can only apply
>for a particular protein, since different proteins seem to stain to differing
>extents (or not at all).

There was a quite interesting paper about the merits of Coomassie
Staining in Electrophoresis (I believe, the 1985 issue; I haven't
the reference handy, though, but could dig it out, if nobody else
finds it).
--
/* Cornelius Krasel, Department of Physiological Chemistry, U Tuebingen    */ 
/* email: krasel@mailserv.zdv.uni-tuebingen.de (Internet)                  */
/*        krasel@chemie.uni-tuebingen.dbp.de (WIN/X400)                    */
/* "People are DNA's way of making more DNA." (anonymous)                  */
From: wetsel_r@wums.wustl.edu
Newsgroups: bionet.molbio.methds-reagnts
Subject: RE: Help with IODO-GEN reagent
Date: 11 Oct 92 14:57:58 GMT
Organization: MIT PLASMA FUSION CENTER
NNTP-Posting-Host: msnews.wustl.edu

Yes... I have extensive experience with Iodogen.  Let's start with simple
things; what are you dissolving it in?  I have never ueDELETE
LASTs
things; what are you dissolving it in?  I have never used anything but
chloroform.  Weigh out what you want and make the necessary dilutions in
chlorform.  Never water - iodogen may not even be soluble in water but water
will all but inactivate it anyway.  To get rid of the chloroform, use a
Pasteur pipette bulb with a needle and with the pipette poking through the
bulb.  This little apparatus is hooked up to the vacuum.  what you basically
want to do is to have your iodogen/clfm (at the concentration you want) under
vacuum as you vortex the solution in the tube.  The vacuum draws off the clfm
leaving the iodogen coated to the glass tubes.  More complex:  iodogen is
water and light sensitive.  Keep the tubes you've made at room temp,
dessicated, and dark (wrapped in foil).  They are only good for 4-6 months if
optimally stored.  The Iodogen reagent itself mustbe kept under similar
conditions.  Look at the bottle as you turn it... does the iodogen reagent
tumble as you turn the bottle, grain by grain?, or does it hold in one position
as you turn it and then fall all at once?  If it does the latter then the
iodogen has taken up water and your labeling efficiency *WILL* be ten-fold
less than what it should be... you need new iodogen.

I hope this helps... if you need any additional information don't hesitate
to call...

David L. Haviland, Ph.D.
haviland@kids.wustl.edu
FAX:314-454-2476
Voice:314-454-6076
From: ULIMJ@CSPUNI12.EARN (Milan Jira)
Subject: Help for recommendation (Specific IgE set)
Original-To: hspnet-l@EARN.ALBNYDH2, medinf-l@EARN.IRLEARN, methods@UK.AC.DARESBURY
Distribution: bionet
Date: Thu, 15 Oct 1992 18:16:12 GMT

 
   Hello networkers,
 
does anybody have a good experience with any (ELISA or strip) test of specific
IgE reliable to (certain extent) diagnose (IN VITRO) food allergy??
 
Fell free to reditribute this request to companies selling these products
giving them my e-mail or Fax number. Unfortunatelly I am still not able to
find reliable representative to consult with him the scientific, technical,
and financial details.
 
        Thanks for your help,
 
Milan Jira, M.D., Ph.D., Head, Dept. of Immunology
Medical Faculty, Charles University
Srobarova 48, 100 42 Praha 10, Czechoslovakia
Fax: +42-2-741075
BITNET: ULIMJ@CSPUNI12    Internet: imunita@earn.cvut.cs
From: story@binah.cc.brandeis.edu
Newsgroups: bionet.molbio.methds-reagnts
Subject: Prime-it Kit
Date: 14 Oct 92 13:09:03 GMT
Reply-To: story@binah.cc.brandeis.edu
Organization: Brandeis University

Dear all:

I slightly goofed in saying that the Prime-it kit was from Promega.  Of course it is from Stratagene.  Sorry for any confusion.

Craig Story
Brandeis U
Waltham, MA
From: jmoon@bcm.tmc.edu (Jon Kenneth Moon)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Help with IODO-GEN reagent
Date: 11 Oct 1992 17:15:07 GMT
Organization: Baylor College of Medicine, Houston, Tx
NNTP-Posting-Host: bcm.tmc.edu
Keywords: Pierce IODO-GEN, plating

I am posting this request for a friend. You can contact him directly by 
phone or FAX or e-mail responses to me and I will forward them. Please let
me know if I should post this to a different group.
	Thanks
	Jon K. Moon at Baylor College of Medicine 
	(jmoon@bcm.tmc.edu)


Message:
	"I'm having trouble with Pierce's IODO-GEN radioiodination reagent.
It's not plating onto the glass tube properly. If anyone has experience
using this reagent please contact me."
			Thanks,
				Joe Henry
				Children's Nutrition Research Center
				Voice: (713) 798-7047 or 798-7069
				FAX: (713) 798-7098
From: ED@micro.uct.ac.za (Ed Rybicki)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Stratagene Quick-Hyb
Date: 12 Oct 92 17:30:36 GMT
Reply-To: ed@micro.uct.ac.za
Distribution: bionet
Original-To: pechan@de.gwdg.dnet.mike, methods@uk.ac.daresbury
X-Mailer: Pegasus Mail v2.1c R5.

> Hi Ed:
> You've said that you've used the PEG 6000 instead dextrane sulfate by hybridiza-
> tions with RNA probes - do you have any experiences by using PEG 6000 in
> Northerns? If yes did you incubate by 68 Celsius? I use everytime:
> -1M NaCl
> -10% dextran sulfate
> -50% formamide
> -salmon sperm DNA.
> Could you send me contents of your Hyb-mix?
>                            Best regards
>                                                    Peter Pechan
>                                                    M.P.I., Goettingen-Germany

Sorry to take so long - student prac marking frenzy time here!

Dr Maud Purves - our RNA queen - tells me she uses the Boehringer-Mannheim
Genius kit buffers without modification for Northerns (occasionally
putting in formamide if she feels it necessary).  She has used PEG-6000
for a blot on my recommendation (Note: I have never used it for RNA
except in the presence of formamide and using 32P at 42degC); it was a dot-
blot for a student practical, of plant viral RNA probed with cloned DIG-
labelled DNA.  She shortened hyb time to 2 hr, and got excellent results
with a 68degC hyb with no formamide.

So:  buffers a la B-M Genius kit (including blocking reagents)
     DIG-labelled probe
     no formamide
     DIG-labelled DNA probe (and my student has just showed me a classical
        chemiluminescent Northern done with DIG-labelled RNA, B-M buffers
        and formamide, no PEG, which looked wonderful)
     up to 10% PEG-6000
     68degC, 1-2 hr hyb
     colour or chemiluminescent detection.

Good luck!
 ____________________________________________________________________
| Ed Rybicki, PhD             |    "Now you've got the hang of it    |
| (ed@micro.uct.ac.za)        | There's nothing you can't do with it |
| Dept Microbiology           |        If you're very into it        |
| University of Cape Town     |         You can't go wrong...."      |
| Private Bag, Rondebosch     |                                      |
| 7700, South Africa          |               -Mad John              |
| fax: 27-21-650 4023         | (Ogden's Nut Gone Flake, Small Faces)|
 --------------------------------------------------------------------
From: FZL39@CSPGCS11.EARN (Ladislav Burysek)
Newsgroups: bionet.molbio.methds-reagnts
Subject: PCR max. lenght
Date: 7 Oct 92 15:40:08 GMT
Distribution: bionet
Original-To: methods@UK.AC.DARESBURY

Hi netters,
 
does anybody know the maximal lenght of the gene amplificable
by PCR? I note, that we want to use it for the transfection
into the eucaryotic cells.
 
Many thanks L.B.
From: mic255z@vaxc.cc.monash.edu.au
Newsgroups: bionet.molbio.methds-reagnts
Subject: Lambda libraries
Date: 13 Oct 92 10:02:36 +1000
Organization: Computer Centre, Monash University, Australia

Dear Netters,

I am interested in using a lambda system to do some primary cloning.
Does anyone have any thoughts on what system is better ie., what are the
relative merits of lambda-ZAP and lambda-DASH compared with EMBL, GEM
or gt11 apart from obvious things like unique cloning sites.

Primarily, I want to clone bacterial outer membrane proteins using specific
antisera to detect recombinants, but I also want to generate lambda libraries
to store for future (unknown) use.  Does this later the comments generated 
by the above question?

ANd finally, lambda-ZAP seems a very good system and I am leaning towards
this.  Has anyone used this system with less than satisfactory results?

Thanks in advance,
Helen Jost,
Department of Microbiology,
Monash University.
From: naffara@crc.ac.uk (Dr. N.A. Affara)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Introns and Exons again...
Date: 7 Oct 92 15:19:36 GMT
Organization: MRC Human Genome Resource Centre

Is there any way of finding out where the intron/exon (exon-exon) 
boundaries are in a cDNA sequence?

I am trying to design PCR primers from a cDNA to work on genomic DNA.
It would help enormously if I could tell where the introns would be.
(Trying to PCR a 50kb+ intron might be a tad difficult! :) )

I have had several replies about max length of introns (200kb?) but not
much about average length of introns and exons.

Thanks very much if you have replied already!

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: bhjelle@carina.unm.edu ()
Subject: Re: Protease inhibitors
Date: Wed, 14 Oct 92 13:36:38 GMT
Organization: University of New Mexico, Albuquerque
Distribution: bionet

In article <9210131601.AA11710@net.bio.net> NANTEL@UNCVX1.BITNET writes:
>
>I'm fishing for information concerning the use of protease inhibitors during
>the purification of MBP-fusion proteins from E.coli. PMSF doesn't seem to
>have any effects on the quality of my protein preps. Furthermore, when I
>use antipain + leupeptin the efficiency of the purification step through
>the amylose column is greatly reduced (7-10 fold lower yields). I feel
>that protease inhibitors are necessary since one of my fusion protein is
>chewed up from 95 to 80 kD during the purification.
>
>I am faily new to the world of protein purification (yes, another molecular
>biologist doing biochemistry without a licence) and any suggestions would
>be most welcome.
>
I could have written this same message. Only the MWs would be
changed to protect the innocent. I'm using trp E fusions and
getting rather severe proteolysis problems. One "solution"
I was considering was to go to pMAL (MBP fusions), but now I'm
not so sure :-).

I am working now on scaring up some protease-deficient E. colis
(lon-, hgpr-) but am told these are pretty tough to work with
(frequent reversions). Will post if this seems to help with my
constructs.

You didn't mention whether it is possible that your fusion is
getting chewed up in the cell, as opposed to during extraction.
I'm pretty sure my problem is intracellular, since I can lyse
the bugs right away, immediately boil in SDS, and see the exact
same degree of proteolysis.

Now I wish I had spent as much time worrying about protein
chemistry as nucleic acids...

Brian
From: chai_z@wehi.edu.au
Newsgroups: bionet.molbio.methds-reagnts
Subject: RE: hybridization problems
Date: 7 Oct 92 05:05:19 GMT
Organization: Walter & Eliza Hall Institute

In article <5OCT92.10175724@wums.wustl.edu>, wetsel_r@wums.wustl.edu writes:
> In a previous article, Klaus.Matthaei@anu.edu.au wrote:
>>>To molecular biologists:
>>>I am having a problem in probing Northern blot membranes ans slot blot 
>>>membranes (Hybond N+). Sllight hazy background is evident on the 
>>>membranes, suggesting that the probe is OK.  However, where the RNA is 
>>>applied in slot blots or where it has migrated to in Northerns appears 
>>>white.  The image looks like a negative.
>>>	If you have encountered this problem or if you know of a 
>>>possible solution, please leave a message here.
>>>Thanks. 
>>>Juan
>> 
>> 
>>G'Day Juan
>> 
>>You have a common problem of not blocking your membrane against
>>non-specific hybridisation.  Nylon membrane needs better blocking than
>>nitrocellulose.  The negative image is due to the probe not hybridising to
>>that part of the membrane and because you have no target = no signal.
>> 
>>For a homologous DNA probe try hybing in: 50% deionised formamide, 2 x PE,
>>7% SDS, 1% BSA (0.5mg/ml Salmon Sperm DNA optional) at 50 degrees.  Wash
>>down to 0.2x SSC 1% SDS at 70.
>> 
>>Good Luck..Klaus
>> 
>>--------------------------------------------------------------------------
>>Catherine Shang
>>Gene Targeting
>>The John Curtin School of Medical Research
>>The Australian National University
>>E-mail: Catherine.Shang@anu.edu.au
>> 
>> 
>>--------------------------------------------------------------------------
> 
> Greetings... Normally I would agree with Catherine.  However, my wife
> and I have experienced this problem in our lab too and it is her impression
> that the problem lies with the lot of N+.  We have always used the standard
> hybridization solution (5X SSC,, 0.2%Ficoll 400,0.2% BSA, 0.2%PVP,0.5%pyro,
> and 1% SDS) ala Maniatis (Salmon sperm optional) and this has worked fine
> for us for years...  until we got this one lot of N+ (#13709) and then all
> h*ll broke loose.  As initially described, our bands were shadowed and the
> background on the blot high.  Only when my wife changed lots of N+ and repeated
> the Southern did it work with low background.  
> 
> Catherine's advise is sound; however, have you had this problem before with
> different lots of N+ or is this a new problem?
> 
> Thanks,
> 
> David
> haviland@kids.wustl.edu


I had the problem of high background in a certain period of time. My colleagues
had the same problem in the same time. I tried very hard to look for the cause.
finally, it worked very well after I made a new batch of buffer and change a 
new tube of 32-P dATP. My feeling was the 32-P (bad batch) and the buffer (too
old).
Organization: City University of New York/ University Computer Center
Date: Wednesday, 30 Sep 1992 19:00:51 EDT
From: Elli Wurtzel <ETWLC@CUNYVM.BITNET>
Newsgroups: bionet.molbio.methds-reagnts
Subject: TA Cloning kits

Any recommendations?
From: Ruud Hovius
Newsgroups: bionet.molbio.methds-reagnts
Subject: quipazine, where to buy ?
Date: 15 Oct 92 16:52:31 GMT
Reply-To: hovius@icphp1
Followup-To: bio.sci
Organization: EPFL

hello,

I am desperately looking for a compound called quipazine, a drug targettted at
the serotonine receptor. Has somebody any clue where to
buy this compound ?

Thanks for the answers !

Ruud Hovius
e-mail : hovius@icphp1.epfl.ch
FAX : +41-21-6934111 
Newsgroups: bionet.molbio.methds-reagnts
From: eboye@radium.uio.no (Erik Boye)
Subject: northern with digoxygenin
Organization: University of Oslo
Date: Thu, 8 Oct 1992 13:01:19 GMT


We have also experienced problems with this combination (read: no success).
The Boehringer people had no good suggestions, so we dropped the method.
However, later I have been told that a positively charged membrane is not 
good for the DIG method, and that a neutral membrane (e.g. from Quiagen)
will do the trick. We have not tried that out.

eboye@radium.uio.no
From: MMELAN@LUCY.WELLESLEY.EDU (Melissa Melan)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Protease Inhibitors
Date: 14 Oct 92 17:16:00 GMT
Distribution: bionet


	In response to Brian's reply concerning possible intracellular
proteolysis of fusion proteins:
	I found the article "Minimizing Proteolysis in Escherichia coli: GeneticSolutions"  by Susan Gottesman in Methods in Enzymology Vol.185 pp.119-129
to be most helpful.  Dr. Gottesman has produced several strains of Lon mutants
and is very cooperative in sharing these E.coli strains with others. See the
article for specific genotypes of each strain.  Hope that this is helpful.
===========================================================================
Melissa Melan
Department of Biological Sciences
Wellesley College
Wellesley, MA  02181
(617) 283-3137
============================================================================
P.S.  Dr Gottesman's address is:
	Laboratory of Molecular Biology
	National Cancer Institute
	NIH
	Bethesda, MD  20892   USA
From: adavies@vax.oxford.ac.uk (Anthony Davies)
Newsgroups: bionet.molbio.methds-reagnts
Subject: gentle radioiodination protocols
Date: 12 Oct 92 08:03:07 GMT
Organization: Institute of Virology, Oxford, UK

What is the best protocol around these days for radioiodinating a protein 
_without_ disrupting its function too much (chloramine T, for instance, seems 
too aggressive for this purpose)?

Looking forward to your answers - will summarise etc.... 

________________________________________________________________________________

Anthony Davies                Voice: 865-512361
Institute of Virology         Fax:   865-59962
Mansfield Road                Vax:   adavies@path.vax.ox.uk.ac (preferred)
Oxford OX1 3SR                       adavies@vax.ox.uk.ac (this account)
UK
________________________________________________________________________________
From: lee@arbo.microbiol.uwa.oz.au (Leanne Sammels)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Gel Reader Software
Date: 16 Oct 92 06:58:24 GMT
Organization: University of Western Australia
NNTP-Posting-Host: kun.microbiol.uwa.edu.au

Dear Netters,

Does anyone know of any 3rd party software available
for use with an IBI Gel Reader.

Lee
Newsgroups: bionet.molbio.methds-reagnts
From: angelo@phoenix.Princeton.EDU (Angelo Gunasekera)
Subject: UGA suppressors
Originator: news@nimaster
Organization: Princeton University
Date: Tue, 13 Oct 1992 05:54:15 GMT


Dear Netters,
I have been trying to express proteins in E.coli UGA suppressor strains
Cells harboring the plasmids grow fine and produce a lot of wild type
proteins although I don't see any suppression of UGA.  I have some
specific questions to ask from others who are working with UGA
suppressor strains.  Please email me at Angelo@bigvax.princeton.edu
Thanks in advance
Angelo
From: ccy@po.CWRU.Edu (Cheung C. Yue)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Protein interaction cloning
Date: 7 Oct 1992 21:34:46 GMT
Organization: Case Western Reserve University, Cleveland, OH (USA)
Reply-To: ccy@po.CWRU.Edu (Cheung C. Yue)
NNTP-Posting-Host: slc12.ins.cwru.edu


Quite some time back, I had responded to a post by stating a number
of new techniques have been described to clone genes for proteins 
which interact with other proteins.  A subsequet post requested references.
My desk has been a mess and it's taken me a while to find them, so here
they are (at least a partial list).


Interaction cloning: identification of a helix-loop-helix zipper protein 
that interacts with c-fos.  Science 256:1014; '92.  
     A fusion protein is constructed with the protein of interest linked to 3 
modules:  a) a FLAG sequence for easy purification, b) an enterokinase 
peptidase cleavage site, and c) a HMK (heart muscle kinase) recognition 
site for 32-P labeling.  The fusion protein is then used for screening an ex
pression library.
     
The two-hybrid system: a method to identify and clone genes for proteins 
that interact with a protein of interest.  PNAS 88:9578; '91.
     DNA binding domain of GAL-4 is fused to protein of interest.  A library 
of sequences is then fused to the activation domain of GAL-4.  Functional 
reconstitution of GAL-4 is read out by a reporter gene with GAL-4 binding sites.
     
A contingent replication assay for the detection of protein-protein 
interactions inanimal cells.  PNAS 88:1086; 91.
     Similar to the two-hybrid system.  Has an amplification loop using 
SV40 T antigen cloned behind GAL-4 binding site, and the fusion protein 
cloned behind SV40 ori.  Newly amplified plasmid sequences are DpnI-
resistant due to lack of methylation, and are selected for by DpnI digestion.
     
Direct selectionfor sequences encoding protease of known specificity.  PNAS 
88:5159;'91.
     Still using GAL-4, but now introduces a specific protease target site 
between the DNA binding domain and the activation domain.  If specific 
protease is co-expressed in the same cell, GAL-4 function would be lost, 
which can then be selected for.

ccy@po.cwru.edu

--