HDTenEightyP/NetNews huggingface dataset:
From: cytogen@chmeds.ac.nz (Martin Kennedy)
Newsgroups: bionet.molbio.methds-reagnts
Subject: RE: Long Seq Gels
Date: 26 Jan 93 21:57:12 GMT
Distribution: bionet

In article <2B643AFA@adminbldg.lan1.umanitoba.ca>, GIETZ@bldghsc.lan1.umanitoba.ca writes:
> ------------------------------------------------------------------------------
> 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-

I used to dry my gels onto the plate, in a 65o^ oven, after placing dampened
Whatman 3M strips around all the edges.  Works fine, but you have to soak the
gels to remove them.  Amersham sell rolls of paper for 35S sequencing gels,
called Hyperpaper (Catalog no RPN1804/1805), in rolls of 20cm or 30cm width,
15.8m long; this stuff works just as well as film, but it's not so convenient
for digitizing. 

Cheers,

Martin

Martin A Kennedy (E-mail = cytogen@chmeds.ac.nz)
Cytogenetic and Molecular Oncology Unit
Christchurch School of Medicine
Christchurch, New Zealand
From: brianb@bcm.tmc.edu (Brian J. Baumgartner)
Newsgroups: bionet.molbio.methds-reagnts
Subject: fusion protein, thanks!
Date: 24 Jan 93 18:33:16 GMT
Organization: Baylor College of Medicine, Houston, Tx
NNTP-Posting-Host: bcm.tmc.edu

Thanks to everyone who responded to my inquiry on fusion protein
expression vectors.  They were all very helpful.  If anyone else has any
additional comments they would still be appreciated!

Brian Baumgartner
brianb@mbcr.bcm.tmc.edu
From: kh@litsun17.epfl.ch (Mark S.)
Newsgroups: bionet.molbio.methds-reagnts
Subject: digoxigenin protein detection?
Date: 28 Jan 93 18:15:40 GMT
Reply-To: kh@litsun.epfl.ch
Distribution: bionet
Organization: Ecole Polytechnique Federale de Lausanne

Who has used the new Digoxigenin protein detection kit from Boehringer?
How sensitive is it? Is it reliable and convenient to use?
We would like to detect protein bands containing less than 1 microgram.
Has anyone experience with this?

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From: BEMDAM@rulgl.LeidenUniv.nl (EDWIN TEN DAM)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: insert-positive blue cols
Date: 21 Jan 93 10:43:00 GMT
Distribution: bionet
X-Envelope-To: methods@daresbury.ac.uk
X-Vms-Cc: BEMDAM
Original-To: methods@uk.ac.daresbury
X-Vms-To: IN%"methods@daresbury.ac.uk"

hi there,

i don't know about 500 bp, but i once insert 51 bp in a pUC vector. In com-
bination with the deletion resulting from cutting the polylinker, everything
was in frame. I checked quite some white colonies before i figured out what
was happening (8-() . I can tell you that weird things happen while cloning.
Out of the first 12 blue colonies, 11 were ok.

-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-
             edwin ten dam - bemdam@rulgl.leidenuniv.nl
From: mball@ac.dal.ca
Newsgroups: bionet.molbio.methds-reagnts
Subject: consumer reports for thermal cyclers
Date: 28 Jan 93 15:59:30 GMT
Organization: Dalhousie University, Halifax, Nova Scotia, Canada

We are in the throes of purchasing a new thermal cycler.
Our requirements are large capacity and preferably not
forced air heating because we are radiolabeling the product
in the reaction.  The models under consideration are:
 an MJR PTC-100-96
 a Techne  model unclear but 96 well format and
 a Hybaid OmniGene
I would greatly appreciate comments on these machines.
We are very concerned with the subject of reliability
since we have heard rumors that the MJR peltier device 
is prone to failure.
Is there an FAQ on this subject?

Thanks in advance

Marty Ball
Biology Dept
Dalhousie University

MBALL@AC.DAL.CA
From: toms@fcs260c2.ncifcrf.gov (Tom Schneider)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Looking for non-coli vector
Date: 28 Jan 93 23:01:41 GMT
Distribution: bionet
Organization: Frederick Cancer Research and Development Center

In article <9301272018.AA19221@umailsrv0.UMD.EDU>,
William_D_WARREN@UMAIL.UMD.EDU (ww40) writes:
>>We are looking for a double stranded, closed circular DNA vector
>>that will not replicate in E coli.

Perhaps you could use the Lox/cre system of recombination to create
a plasmid in vivo which cannot replicate?

  Tom Schneider
  National Cancer Institute
  Laboratory of Mathematical Biology
  Frederick, Maryland  21702-1201
  toms@ncifcrf.gov
From: brianb@bcm.tmc.edu (Brian J. Baumgartner)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Northern
Summary: RE: Northern
Date: 24 Jan 93 19:11:46 GMT
Distribution: bionet
Organization: Baylor College of Medicine, Houston, Tx
NNTP-Posting-Host: bcm.tmc.edu

In article <9301231934.AA10759@net.bio.net>, GWU5042@GWUVM.GWU.EDU (Hans Xie) writes:
> Has anyone had exprience in mixing up two probes in one northern, such
> as one probe for detecting signal, another actin probe for normalization?
> How is it as compared to doing northern twice with stripping?


Make sure there is no complementarity between the two probes.  If you are use-
ing riboprobes remember that short stretches of the vector sequence are
included in the RNA probe which may be complementary. This precaution will
prevent hybridization between your two probes instead of to your targets. 
Also make sure that the probes themselves will not cross-hybridize to
target and actin mRNA. The presence of multiple un-related probes in solution
should not otherwise influence the kinetics of hybridization to
filter-bound templates, since the probe is included in excess anyway.  The
only other caveat that I can think of is to make sure that your target and
actin mRNA do not co-migrate on the gel!  It might be worth an initial
experiment to check for cross-hybridization of your two probes by setting
up duplicate blots with one or the other probe; you may have to adjust
stringency conditions to ensure specific hybridization.

Good luck!

Brian Baumgartner
From: Mark S. Rose <msr2@cornell.edu>
Newsgroups: bionet.molbio.methds-reagnts
Subject: Fungal Transformation
Date: 23 Jan 1993 16:51:50 GMT
Organization: Cornell University
Distribution: world
NNTP-Posting-Host: cu-dialup-0311.cit.cornell.edu
X-UserAgent: Nuntius v1.1.1d7
X-XXDate: Sat, 23 Jan 93 12:00:13 GMT


msr2@cornell.edu
Dept. of Plant Pathology, Cornell U.
Ithaca, NY, USA
From: preissj@CLVAX1.CL.MSU.EDU ("J Preiss--Seq Anal")
Newsgroups: bionet.molbio.methds-reagnts
Subject: re hybridization problems
Date: 27 Jan 93 18:39:00 GMT
Distribution: bionet

Hi Chriss

	Well, you did not give very much information in your request for help.
Since I really can't trouble shoot your method very well, I will just give you
mine.  I use 1.5x SSPE, 1.0% SDS, and 0.5% blotto (Carnation instant milk, 
made fresh) for my hybe and prehybe for both northern and southern work.
That's it.  Nothing else.  I used to use sheard DNA, but have since left that
out with no ill effect.  I specifically do not like any of the "hybridization
enhancers" like denharts, formamide, and dextran.  I find all of them to give
background problems in genomic southerns where it counts most.  I hybredize
at 55 to 65 degrees.  Tm is defined by the following formula in this mix:

Tm=16.6(log[Na+])+0.41(%GC)+81.5+500/(probe length bp)

[Na+] = 0.15 for 1x SSC		log 0.15 = -0.824
      = 0.015 for 0.1x SSC	log 0.015 = -1.824

probe length from standard oligonucleotide procedures = about 250 bp 
	(regardless of the template length)

If GC is assumed to be about 50%, then:
	Tm = 86 C	in 1.0x SSC
	   = 70 C 	in 0.1x SSC

	These numbers are for the wash conditions, which I do in 0.1% to 
1.0% SDS and 0.1x SSC to 1.0x SSC at 55 C.  I find that going below 55
gives higher background, while changing salt gives varried stringency 
without background.

	Good Luck and feel free to write back for more info.

		Dr. Leonard N. Bloksberg
		PreissJ@clvax1.cl.msu.edu
		Dept. of Biochemistry
		Michigan State University
From: bi__mvw@SELWAY.UMT.EDU (Michael van Waes)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: RNA gels
Date: 26 Jan 93 16:44:37 GMT
Distribution: bionet

>
> Does anyone know what size RNA correspond to the
>bromophenol blue dye front?
>
>-fm
>
It depends on the % polyacrylamide; in general (this is VERY approximate and
variable): 

	%	BP Blue		XCyanol
	5	30		120
	20	5		15-20


I insist this is VERY unreliable, and a MW marker is highly recommended (you
can always label it to see it in the autorad).

Cheers..... Michael van Waes.
From: BMA14432@VAX1.UTULSA.EDU
Newsgroups: bionet.molbio.methds-reagnts
Subject: RE: Shuttle vector- Hirt lysate yields
Date: 24 Jan 93 14:13:51 GMT
Distribution: bionet


Britt,
I have found when performing the Hirt procedure it is very important
to GENTLY rock the tube(I do these in eppendorff tubes) after adding
the SDS. I have also found lysis to be improved if one warms the 
tube during this process(I hold it over a bunsenburner...not too close!:)).
I also use library competent cells(MC1061/P3 from Invitrogen). Hope that
Helps.

Mark Brudnak
University of Tulsa
BMA14432@VAX1.UTULSA.EDU
From: aescalan@ORION.OAC.UCI.EDU (Ananias Escalante)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Problems cloning PCR products
Date: 25 Jan 93 23:03:05 GMT
Distribution: bionet


Hello!

I am interesting in cloning PCR products. Does somebody know a good reference?

				Ananias Escalante

				aescalan@orion.oac.uci.edu
From: GIETZ@bldghsc.lan1.umanitoba.ca
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re:RNA gels
Date: 26 Jan 93 17:54:00 GMT
Distribution: bionet

-----------------------------------------------------------------------------
>I am about to do a Northern blot.  I am thinking of running the
>RNA out on 1% formaldehyde gel.  The fragment I want to detect
>is at least 3 kb.  The question is how long should I run the
>gel?  Does anyone know what size RNA correspond to the
>bromophenol blue dye front?

>-fm
------------------------------------------------------------------------------
---
I used to run 1.5% agarose: formaldehyde gels and 
that size of mRNA ran  above the Xylene Cyanol marker.
If you run the gel so that the bromophenol blue is just at the end
the XC will be about mid way in a 1.5% agarose gel.  Hope this 
helps! I never ran 1% agarose:formaldehyde because of the resolution
needed.
________________________________________
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: DAC050@MUVMS3.MU.WVNET.EDU
Newsgroups: bionet.molbio.methds-reagnts
Subject: sequence of psg5 vector...
Date: 28 Jan 93 20:17:00 GMT
Distribution: bionet

HI Net fellows:

Does anyone has a pSG5 vector sequence or restriction map. If you have, please
email the sequence of pSG5 vector.

Thanks in advnace.

Dinakar
From: GWU5042@GWUVM.GWU.EDU (Hans Xie)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Northern
Date: 23 Jan 93 18:31:17 GMT
Distribution: bionet

Has anyone had exprience in mixing up two probes in one northern, such
as one probe for detecting signal, another actin probe for normalization?
How is it as compared to doing northern twice with stripping?
From: wsun@jeeves.ucsd.edu (Fiberman)
Newsgroups: bionet.molbio.methds-reagnts
Subject: RNA gels
Date: 25 Jan 93 22:24:26 GMT
Organization: University of California, San Diego

I am about to do a Northern blot.  I am thinking of running the
RNA out on 1% formaldehyde gel.  The fragment I want to detect
is at least 3 kb.  The question is how long should I run the
gel?  Does anyone know what size RNA correspond to the
bromophenol blue dye front?

-fm
From: FORNACE@FCRFV2.NCIFCRF.GOV
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: problems with mrna quantitation
Date: 26 Jan 93 22:36:13 GMT
Distribution: bionet

>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


try: 
Hollander, M.C. and Fornace, A.J., Jr.:  Estimation of relative mRNA content by
filter hybridization to a polythymidylate probe.  Biotechniques 9:  174-179,
1990.
From: B7JP@MUSICT.MCGILL.CA (B7JP)
Newsgroups: bionet.molbio.methds-reagnts
Subject: DNA PURITY?
Date: 28 Jan 93 22:34:10 GMT
Organization: McGill University

Dear researcher:

Does anybody know for a DNA solution (after
phenol/chloroform/isoamylalcohol extraction, precipitation with
ammonium acetate and ethanol, rinsing in ethanol 70% and dissolving in
TE) what does a ratio of A260nm/A280nm between 2 to 4 mean?

Thanking you in advance!

Seyed
From: A8101DAL@vm.univie.ac.at (Martin Hetzer)
Newsgroups: bionet.molbio.methds-reagnts
Subject: photography
Date: 28 Jan 93 14:38:26 GMT
Organization: University of Vienna

Usually people use the POLAROID system to take pictures of their
EB stained gels. Who has used reflex cameras for this purpose
and wich film type is recommended?
Thanks in advance
Martin
From: JCHEN@MACC.WISC.EDU (Jianchi Chen)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Long Seq Gels
Date: 29 Jan 93 03:48:00 GMT
Distribution: bionet


 
> Also, we don't have a gel dryer that accomodates long gels.  Any
> alternative drying method?
 
>>I bind the gel to one of the glass plates, making for easier handling, but
>>meaning a regular gel dryer can't be used.  I now put the plate in an oven
>>for an hour at about 100 deg C.  Before we got a large oven I used to put
>>the gel under a group of hair dryers for a couple of hours.  It was noisy
>>and not as fast as an oven, but it works.  I would think this would also
>>work for a gel transfered to paper.
 
Yes! A gel transfered to paper works better.  What I usually do is: after
transferring the gel to Whatman paper, clamp it on a piece of metal board with
the gel side up, dry the gel in an oven.  It may work even faster to dry a
gel this way with a heater under a hood.  By the way, two pieces of metal
board clamped together can be a cassette.
 
Jianchi Chen
Dept. of Food Microbiology and Toxicology
Univ. of Wisconsin
JCHEN@MACC.WISC.EDU
From: cytogen@chmeds.ac.nz (Martin Kennedy)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Northern
Date: 24 Jan 93 22:34:41 GMT
Distribution: bionet

In article <9301231934.AA10759@net.bio.net>, GWU5042@GWUVM.GWU.EDU (Hans Xie) writes:

> Has anyone had exprience in mixing up two probes in one northern, such
> as one probe for detecting signal, another actin probe for normalization?
> How is it as compared to doing northern twice with stripping?
-- 

I don't think it would be wise to mix the actin probe with your test probe
unless you have an idea of the level of expression relative to actin.  I always
do my test probing first, then reprobe with actin without bothering to strip
off the first signal. 

Cheers,

Martin

Martin A Kennedy (E-mail = cytogen@chmeds.ac.nz)
Cytogenetic and Molecular Oncology Unit
Christchurch School of Medicine
Christchurch, New Zealand
From: pnh@fcsparc6.ncifcrf.gov (Paul N Hengen)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Problems cloning PCR products
Summary: Check the FAQ list for references
Keywords: FAQ list
Date: 27 Jan 93 16:50:21 GMT
Distribution: bionet
Organization: Frederick Cancer Research and Development Center

aescalan@ORION.OAC.UCI.EDU (Ananias Escalante) writes:

>I am interesting in cloning PCR products. Does somebody know a good reference?

You might want to check the FAQ list for PCR references. There will be
a newly updated FAQ list posted here soon if you haven't seen it.
You can also get the old version by anonymous ftp to ncifcrf.gov
Simply get the file pub/methods/FAQlist

Paul N. Hengen
National Cancer Institute
Frederick Cancer Research and Development Center
Frederick, Maryland 21702-1201 USA
From: eric@bcserv.wustl.edu (Hugo (Eric))
Newsgroups: bionet.molbio.methds-reagnts
Subject: Chicken Dihydrofolate Reductase cDNA clone Needed
Date: 23 Jan 93 15:29:24 GMT
Organization: Washington University in Saint Louis, MO USA
Distribution: na
NNTP-Posting-Host: bcserv.wustl.edu
Summary: Does anyone out there have a chicken DHFR cDNA clone to share?
Keywords: chicken, DHFR

Dear Bionetters,
     I am in search of a chicken DHFR clone.  I would like to express
the chicken dihydrofolate reductase protein in E. coli and in need
of a cDNA for this enzyme.  If anyone out there has such a beast and
is willing to part with a sample I would greatly appreciate it.
The cDNA does not necessarily need to be in an expression vector as
subcloning is not a problem (making the cDNA is a project I'd rather
avoid however).  Any pointers would be greatly appreciated.  Please email
any resposes to the address below:                Thanks,        Eric ;-)
                    eric@bcserv.wustl.edu
-- 
  Eric R. Hugo, Postdoctoral Research Associate |eric@bcserv.wustl.edu
  Dept. of Biochemistry and Molecular Biophysics|                
  Washington University School of Medicine      |               
  Box 8231, St. Louis, MO 63110_________________| (314) 362-3342
Newsgroups: bionet.molbio.methds-reagnts
From: mike@medsun.unige.ch (michel morris)
Subject: ABI sequencer: reaction clean-up ??
Organization: University of Geneva, Switzerland
Date: Fri, 22 Jan 1993 12:29:27 GMT

	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
From: Mark S. Rose <msr2@cornell.edu>
Newsgroups: bionet.molbio.methds-reagnts
Subject: Transforming Fungi
Date: 28 Jan 1993 00:27:35 GMT
Organization: Cornell University
Distribution: world
NNTP-Posting-Host: cu-dialup-0118.cit.cornell.edu
X-UserAgent: Nuntius v1.1.1d7
X-XXDate: Wed, 27 Jan 93 19:36:09 GMT

Has anyone attempted to make protoplasts from or transform Mycosphaerella
zeae-maydis (anamorph: Phyllosticta maydis)?
Also, does anyone have  a cloned promoter fom any Mycosphaerella spp.
that they would be willing to share?
Thanks
Mark
msr2@cornell.edu
Dept. of Plant Pathology, Cornell U.
Ithaca, NY, USA
From: biotec@psl.wisc.edu
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Direct Lambda Sequencing : new answers to old thread ?
Date: 26 Jan 93 16:26:57 GMT
Reply-To: biotec@psl.wisc.edu
Organization: University of Wisconsin - Physical Science Lab

Promega has a system for sequencing directly from lambda DNA using sequencing grade Taq DNA polymerase called FMol DNA Sequencing System. I can give readable sequence from 130ng (4fmoles)of lambda using a P32 end labeled primer. It is not possible to sequence lambda with S35 internal incorporation using this system.At the time we discontinued selling our T7 DNA Polymerase Sequencing System we were not able to get reproducable sequence from lambda.
Angela Ryan
Technical Services
Newsgroups: bionet.molbio.methds-reagnts
From: jm68@quads.uchicago.edu (James R. Mensch Jr.)
Subject: Polyclonal screening bkg.
Reply-To: jm68@midway.uchicago.edu
Organization: University of Chicago Computing Organizations
Distribution: na
Date: Mon, 25 Jan 1993 23:45:05 GMT

We are having a problem with high background in our attempts to screen a
lambda ZAPII cDNA library.  The instructions accompanying the library, the
protocol obtained with the screening/detection kit from the same source as the
library as well as that from another vendor, and the technical services people
have all been consulted to no avail.  Though we are not rookies at this game,
perhaps there is some good trick we haven't heard of yet.  The parameters of
our system are these:
- A lambda ZAP-II rat liver cDNA library is plated on XL1-Blue bacteria
- IPTG-saturated (10 mM) nitrocellulose filters, S&S BA85,are used to prepare
  plaque lifts (2 sets).
- Rinsing/washing of filters is done in 'TNT'(10 mM Tris/150mM NaCl/
  0.05% Tween 20, pH 8.0); blocking in TNT with 6% BSA added.
- Primary antibody is a rabbit polyclonal. The preparation in use was 'stripped'  of species reactive to E.coli/phage proteins by incubation with filters
  bearing proteins from a bacterial/phage lysate.
- Secondary antibody is goat anti-rabbit with conjugated alkaline phosphatase,
  signal production is by incubation with BCIP/NBT.
Prior to use of the stripping lysate, an extremely heavy background was obtainedon all primary round filters.  Post-stripping this background was reduced to a
light violet tinting with non-positive (ie. all) plaque images discernable.
Over time this preparation has yielded progressively heavier background to the
point where the plaque images are now seen as lighter spots!  Negative negativesif you will.  These effects are independent of reagent batches, solutions, etc.
etc..  We are contemplating adding an intermediate antibody to the system.
We have used a monoclonal to screen filters from the same filter lot lifted 
from plates of the same batches as used with the polyclonal, the monoclonal gives no background problems albeit using a HRP-conjugated anti-mouse 2nd Ab.
No definite positives yet either!  A positive control phage (globin cDNA insert,
but detected by a monoclonal recognizing the B-gal portion of the fusion proteinproduct) gives a good signal against a field of negative (gt10) plaques.  Thus
the polyclonal Ab prep appears to be the culprit, yet it works well with West-
erns as regards binding to the target protein.
Any ideas for us?  Thanks in advance for your consideration.  Jim M.
<j-mensch@midway.uchicago.edu>
-- 
===============================================================================
Jim Mensch : j-mensch@uchicago.edu
             menche@biovax.uchicago.edu
===============================================================================