HDTenEightyP/NetNews huggingface dataset:
From: dknudson@OPS.AGSCI.COLOSTATE.EDU
Newsgroups: bionet.molbio.methds-reagnts
Subject: RE: Tms refs
Date: 26 Aug 92 14:29:37 GMT
Distribution: bionet


To: MURPHY@UK.AC.AFRC.JII
Subject: Re: TM
Newsgroups: bionet.molbio.methds-reagnts
Organization: Colorado State University, Fort Collins, CO  80523
Cc: 
Bcc: 

In article <1992Aug26.111127.1072@gserv1.dl.ac.uk> you write:
>Dear Netters,
>I would be grateful for any information you can give me about calculating the
>Tm's for oligo hybridization. My problem is this:
>Sambrook/Fritsch/Maniatis (whose endeavours I have been grateful for on many
>occasions) quote three equations to calculate Tm's (Vol2, p9.51). As has been 
>pointed out before, the value for log[Na] should be added, not subtracted in
>the DNA/DNA equation. Nobodys perfect (:^). My interest is in the DNA/RNA and 
>RNA/RNA equations.
>1. Where %G+C is mentioned, then I assume 60%G+C is 0.6 and not 60 (which 
>isn't %G+C in my book)?
>2. Can anybody provide me with references for these equations? I have looked
>at the ones quoted in Sambrook et al. but I cannot find the equations in these
>papers. Thanks in advance.
>Cheers,
>George Murphy
>*******************************************************************************
>George Murphy                     |Snailmail: 
>                                  |     Molecular Genetics,
>JANET:murphy@uk.ac.afrc.jii       |     Cambridge Laboratory,
>INTERNET:murphy@jii.afrc.ac.uk    |     JI Centre for Plant Science Research,
>                                  |     Colney Lane,
>Telephone: +44 603 52571          |     Norwich NR4 7UJ,
>Fax:       +44 603 505725         |     U.K.
>*******************************************************************************
RNA/RNA Tms = Bodkin and Knudson Virology 143:55-62(85); Wahl etal Meth.Enzymol
              152:399-407(87).
DNA/RNA Tms = Birnstiel etal J. Mol. Biol. 63:21-29(72); Wahl etal Meth.Enzymol
              152:399-407(87).
DNA/DNA Tms = There are 3 equations.
              1) McConaughy etal Biochem 8:3289; Wahl etal Meth.Enzymol 152:399
                 -407(87).
              2) Wahl etal Meth.Enzymol 152:399-407(87).
              3) Baldino etal Meth.Enzymol 168:761-777(89).
Hope these help!  Please let know if you find any discrepancies.

=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=
Dr. Dennis L. Knudson, Professor of Entomology and Microbiology
Department of Entomology
College of Agricultural Sciences    Telephone: (303) 491-7255
Colorado State University                 Fax: (303) 491-0564
Fort Collins, CO  80523  USA         Internet: dknudson@ops.agsci.colostate.edu
=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=*=

Newsgroups: bionet.molbio.methds-reagnts
From: pcj1@cunixf.cc.columbia.edu (Pierre Jelenc)
Subject: semi-log paper
Reply-To: pcj1@cunixf.cc.columbia.edu (Pierre Jelenc)
Organization: Columbia University
Distribution: usa
Date: Wed, 26 Aug 1992 00:04:20 GMT


Has anyone had trouble getting semi-log graph paper recently? We were told
by the manufacturers and various university bookstores that it has been
discontinued! Did our secretary misunderstand something, or is there some
dark design behind this all?

Pierre

Pierre Jelenc                        pcj1@cunixf.cc.columbia.edu 
                                    Columbia University, New York
From: afc@gnv.ifas.ufl.edu
Newsgroups: bionet.molbio.methds-reagnts
Subject: DNA labelling kits
Date: 4 Sep 92 11:53:59 -0500

A few years ago we switched from labelling by nick translation to
labelling by random priming.  We use BRL kits for both.  Besides 
being easier to use and more reliable, random priming gives about
100x higher specific activity.   Mostly we are probing total cell
DNA (from single mosquitoes) for mitochondrial or ribosomal genes, 
so probe should be in excess.

My question is: why don't we see a 100x increase in hybridization
intensity?  RP probes give a little better results than NT probes,
maybe 2x or 3x in our hands.  

Dextran sulfate doesn't help much, so it doesn't seem to be a cot 
problem due to low probe concentration.

We use the probes the same day that we make them.  Incorporation is
about one 32P / 30 bases.  I don't think that strand breakage due to
radiodecay is the answer.

I am guessing that probe length might be a problem: NT probes have a
several hundred bases of unlabelled DNA with the labelled DNA
attached.  RP probes have six unlabelled bases attached to the label.
Depending on how long the synthesized strands are, this could make a
big difference.  If so, would playing with primer concentration help?
What about other labelling kits?

Any suggestions or comments would be appreciated.

Andrew Cockburn
USDA
From: saparici@crc.ac.uk (Dr. S.A.J.R. Aparicio)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: PCR buffers
Date: 3 Sep 92 11:01:24 GMT
Reply-To: saparici@crc.ac.uk (Dr. S.A.J.R. Aparicio)
Organization: University of Cambridge, Department of Medicine


In article <loryn.5.0@microbiol.uwa.oz.au>, loryn@microbiol.uwa.oz.au (Loryn Sellner) writes:
> We have recently changed our Taq polymerase supplier, and have run into a 
> few problems deciding what reaction buffer to use. The left over 10x buffer 
> from our old Taq source seems to be giving a lot of background non-specific 
> product when used with the new Taq, which we never saw with the old Taq. 
> When we use the new buffer with the new Taq, we get no background, but we 
> also see a ten fold drop in sensitivity. The major differences between the 
> old and new buffer are that the old buffer contained ammonium sulphate as a 
> salt rather than potassium chloride, and also contained gelatin and 2-
> mercaptoethanol, whereas the new buffer does not have these but does 
> have Triton X-100. I would be interested in hearing:
> 1) any ideas as to why we are getting these results
> 2) anybody else who has encountered problems in using buffers
> 3) anybody else who has their own recipe for making PCR buffer

Loryn, you don't say which suppliers.
I have certainly experienced sensitivity difference in my hands between Cetus Taq and 
Promega Taq. Although we have found the Promega Taq and buffer to be good at times, we have
also experienced sufficient variation so that we no longer use it for sensitive applications.

The buffer we use with Cetus Taq contains only Tris, KCl and MgCl2 - no gelatin, Triton or
anything else, this seems to have given the cleanest and most consistent results.

WE/I have no affiliation at all to any of these companies, so all the usual disclaimers
apply.

yours

Sam Aparicio
MRC Molecular Genetics Unit
Cambridge
CB2 2QH
From: toms@fcs260c2.ncifcrf.gov (Tom Schneider)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: semi-log paper
Date: 26 Aug 92 21:35:21 GMT
Distribution: usa
Organization: Frederick Cancer Research and Development Center

In article <1992Aug26.000420.6359@news.columbia.edu> pcj1@cunixf.cc.columbia.edu (Pierre Jelenc) writes:

>Has anyone had trouble getting semi-log graph paper recently?

I stopped using graph paper years ago because I wrote a program called xyplo
which can do this (and a few other nice x-y plot graphics).  It's hard to use
but rather powerful and will transport to any computer that has a reasonable
Pascal or C compiler.  The output is in PostScript.  It's available by
anonymous ftp from ncifcrf.gov in directory pub/delila.  Get the xy* files.

  Tom Schneider
  National Cancer Institute
  Laboratory of Mathematical Biology
  Frederick, Maryland  21702-1201
  toms@ncifcrf.gov
From: TY9283@UACSC2.ALBANY.EDU (Jen-Wei)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Oligosaccharides
Date: 2 Sep 92 21:49:44 GMT
Distribution: bionet

Hi,

   Does anyone know the phone or FAX # for this company, Chembiomed
Synsorb?

   Are there anyother companies produce the following compounds?

      H type-1 oligosaccharide
      H type-2 oligosaccharide
      Lacto-N-biose I
      N-acetyl-2'-O-methyllactosamine

                                                      Thanks in advance

                                                              Jen-Wei
From: ANSIDHP@EARN.OSUCC
Newsgroups: bionet.molbio.methds-reagnts
Subject: PCR with E. Coli
Date: 4 Sep 92 12:44:00 GMT
Distribution: bionet
Original-To: methods@UK.AC.DARESBURY

Hello Netters,
I routinely perform PCR to amplify short (100-500) bp regions
of mammalian DNA.
I am now about to help a colleague attempt to amplify a large
(%2 kb) fragment in E. Coli.
For mammalian DNA I normally use:
100 ng genomic template
300 nM each primer
200 uM each dNTP
1.5 mM MgCl
in 50 ul reactions.
Should I alter any of these conditions for amplification in
E. Coli? I would think that the concentration of genomic template
might be reduced, due to the smaller genome size.
Also, are there some tricks for amplifying longer regions of DNA?
I normally use 1 minute time spans for all 3 steps in a cycle.
Should the extension interval be lengthened?
Any helpful comments would be greatly appreciated!
Daniel Pomp
Department of Animal Science
Oklahoma State University
ANSIDHP@OSUCC
(405)744-6070 (Tel)
(405)744-5747 (Fax)
From: URA1335@FRCITI51.BITNET
Newsgroups: bionet.molbio.methds-reagnts
Subject: estimated amount of testis-specific mRNA?
Date: 29 Aug 92 13:09:01 GMT
Distribution: bionet

Hi! Netters,
I have a theoratical question:
Does anyone of you know or have any reference
about the estimated amount of mRNA specifically
expressed in testis; did somebody calculated
it? say, 10,000; 20,000 ;more? hox much??
we knew that testis as organ is a higly
specialised assembly of tissues...
For exemple what is this amount compared
the one for brain or liver??
Thank you in advence.
Dr. Serdar ARAL
E-Mail: "ura1335@frciti51.bitnet"
Newsgroups: bionet.molbio.methds-reagnts
From: loryn@microbiol.uwa.oz.au (Loryn Sellner)
Subject: PCR buffers
Organization: Dept. Microbiology
Date: Thu, 3 Sep 1992 06:08:21 GMT

We have recently changed our Taq polymerase supplier, and have run into a 
few problems deciding what reaction buffer to use. The left over 10x buffer 
from our old Taq source seems to be giving a lot of background non-specific 
product when used with the new Taq, which we never saw with the old Taq. 
When we use the new buffer with the new Taq, we get no background, but we 
also see a ten fold drop in sensitivity. The major differences between the 
old and new buffer are that the old buffer contained ammonium sulphate as a 
salt rather than potassium chloride, and also contained gelatin and 2-
mercaptoethanol, whereas the new buffer does not have these but does 
have Triton X-100. I would be interested in hearing:
1) any ideas as to why we are getting these results
2) anybody else who has encountered problems in using buffers
3) anybody else who has their own recipe for making PCR buffer

Much appreciated.



Loryn Sellner
Department of Microbiology
From: "michael coyne" <michael.coyne@channel1.com>
Newsgroups: bionet.molbio.methds-reagnts
Subject: fin i enzyme info?
Date: 1 Sep 92 22:52:17 EST
Reply-To: "michael coyne" <michael.coyne@channel1.com>
Distribution: bionet
Organization: Channel 1 Communications


A colleauge of mine at Harvard asked me if I would post a request for
*any* information anyone might have on a restriction enzyme known as Fin
I.  We know its recognition sequence is GTCCC, but that's about it.  She
has contacted every commercial supplier of enzymes we know of, but no
one seems to carry it, though it is listed in some of the catalogs in
the appendix sections.

Would anyone know where we might get this enzyme (or an isoschizomer)?
Failing that, would anyone know the primary reference for this enzyme?
And, failing that, does anyone know what organism Fin refers to?

As is readily apparent, we know next to nothing about this enzyme except
it's recognition sequence, and would appreciate any information at all.

Thanks...

Michael Coyne
michael.coyne@channel1.com
---
  SLMR 2.1a  Backup not found: (A)bort (R)etry (P)anic
--
Channel 1 (R)   Cambridge, MA
From: coyne@uctvax.uct.ac.za
Newsgroups: bionet.molbio.methds-reagnts
Subject: X-GAL CLEAVAGE BY V. CHOLERAE
Date: 1 Sep 92 15:21:33 +0200
Organization: University of Cape Town

Dear Netters,

I am attempting to isolate transposon generated lacZ operon fusions in 
Vibrio cholerae by cleavage of X-gal to generate blue colonies.  According to
Bergeys, V. cholerae is lactose negative.  However, I have found that
V. cholerae is able to cleave X-gal to generate blue colour which is a major 
problem with respect to my strategy.  I would greatly appreciate it if anyone 
could supply me with an answer to the following question:

   Does V. choerae possesses a beta-galactosidase enzyme?
   (The bacterium was scored as lac- as a function of growth on lactose).

You may post the answer e-mail it directly to me.

Many thanks in advance,

Vernon Coyne
vernon@micro.uct.ac.za
From: tyersome@toxic (Randall Tyers)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Strange receptor binding studies, or are they?
Date: 26 Aug 1992 17:00:22 GMT
Organization: Plant Biology
NNTP-Posting-Host: toxic.berkeley.edu

In article <1992Aug26.134234.1@vaxa.strath.ac.uk> cdds01@vaxa.strath.ac.uk writes:
>I have recently been doing some receptor-binding studies
...
>The thing is the counts I get back show that tracer binding
>increases as I add cold competitor (instead of decreasing as I expected).
>Does anyone
 have any ideas on why this should happen?
>
>Thanks in advance
>Pete Crilly

If your receptor binds cooperatively then you may need to add a critical 
concentration of ligand before a significant amount of binding occurs.
If this is responsible for your observation then you should be able to get 
competition by adding even more competitor.

Hope this helps.

Randall Tyers
Newsgroups: bionet.molbio.methds-reagnts
From: ccy@po.CWRU.Edu (Cheung C. Yue)
Subject: Re: Protein-protein interactions
Reply-To: ccy@po.CWRU.Edu (Cheung C. Yue)
Organization: Case Western Reserve University, Cleveland, OH (USA)
Date: Wed, 26 Aug 92 10:10:44 GMT


In a previous article, zxmkr08@mailserv.zdv.uni-tuebingen.de (Cornelius Krasel) says:

>This may be a fairly general question for bionet.molbio.methds-reagnts,
>but anyways... what kind of methods do exist for studying interactions
>between proteins, more specifically, to find proteins which interact with
>a known protein? So far, I am aware of crosslinking methods, followed by
>immunoprecipitation.


Yet another method is expression cloning.  An expression library is probed
with a labelled protein (or peptide domain of a protein) to search for
cDNA clones expressing an epitope which binds/interacts with the protein
probe.  That way, you get the gene for the interacting protein; thereby
saving enormous efforts in protein isolation, purification and sequencing.
A number of successful applications have appeared in the literature in 
the recent past.

ccy@po.cwru.edu
-- 
Newsgroups: bionet.molbio.methds-reagnts
From: ca566@cleveland.Freenet.Edu (Michael Holloway)
Subject: No more free 33P from NEN
Reply-To: ca566@cleveland.Freenet.Edu (Michael Holloway)
Organization: Case Western Reserve University, Cleveland, OH (USA)
Date: Mon, 31 Aug 92 20:58:42 GMT


Sorry.  I posted a message about getting a sample of alpha 33P dATP from
NEN and have just been informed that they've stopped the free offer.  They
had been offering it just weeks ago but it looks like the free ride is
over.
-- 
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: bcrosby@pbi.lan.nrc.ca
Newsgroups: bionet.molbio.methds-reagnts
Subject: References on Amino Acids,
Date: 3 Sep 92 15:30:00 GMT
Distribution: bionet

Dear Dr. Last,

My lab has been investigating the biochemistry of ALS enzymes in B. napus, 
with a lesser emphasis on A. thaliana for comparative purposes. Given that 
emphasis, would the B. napus material be of use to you? We have recently 
submitted a manuscript to Plant Physiol, and hope to see it published later 
this year.

Bill Crosby
From: agoodrid@vaxa.weeg.uiowa.edu (Stephen Klautky)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Luciferase activity by scintillation counting?
Date: 3 Sep 92 17:33:00 GMT
Organization: University of Iowa, Iowa City, IA, USA
News-Software: VAX/VMS VNEWS 1.41

Hi,

Has anyone tried to use the scintillation counter to measure
luciferase activity?  I am interested in using this method to
assay luciferase from transfected cell extracts.

Is it as sensitive as using a luminometer?  Reproducible?
Is it linear of a large range of enzyme activities?

The advantage, as I understand it, of the scintillation method is
that it costs much less because one does not have to invest in a
luminometer.

Can you point me to references on this topic?

Thanks,

-=] Stephen Klautky [=-     AGOODRID@VAXA.WEEG.UIOWA.EDU
From: BMHTLAI@CCVAX.AS.EDU.TW
Newsgroups: bionet.molbio.methds-reagnts
Subject: Purification after thrombin or Factor Xa cleavage
Date: 28 Aug 92 03:43:00 GMT
Distribution: bionet

Dear netters:

Can anyone of you point out a simple method to remove proteases, in 
specifically thrombin and Factor Xa?
I have known there is an affinity column (benzamidine-sepharose 6B) for removal
of thrombin, but I have not got the protocol yet. Can someone comment on it
, too?

Thanks in advance.

H.-T. Lai
bmhtlai@ccvax.as.edu.tw
From: camdna@ubvmsb.cc.buffalo.edu (Darren Natale)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Partial digestions with ApaL1 - help!!
Date: 1 Sep 92 16:27:00 GMT
Organization: University at Buffalo
News-Software: VAX/VMS VNEWS 1.41

In article <180321INNc2c@usenet.INS.CWRU.Edu>, axa12@po.CWRU.Edu (Ashok Aiyar) writes...
> 
>I'm doing partial digestions on a plasmid that has 4 ApaL1
>sites, and can't get the partials to work.  I have tried to
>dilute the enzyme over 2 logs and independently have also tried
>timed digestions.  I seem to get the enzyme cutting at all four
>sites or none of the sites.
> 
I have not tried partials with this particular enzyme, but have done others.
When confronted with the situation described, I have tried doing the digests
under sub-optimal conditions (for example, a different salt concentration in
the buffer, etc.) The easiest parameter to vary is temperature. I have been
successful using low temperature digestions (ie, 15-23 degrees Celsius).
This may work for you. Good Luck.

DN
From: skrshaw@uk.ac.reading.susssys1
Newsgroups: bionet.molbio.methds-reagnts
Subject: N2 storage of murine cell-lines
Date: 26 Aug 92 19:20:52 GMT
Distribution: bionet
Original-To: methods@uk.ac.daresbury

I've had problems in successfully freezing macrophage cell cultures so they 
can be successfully resuscitated. The problem does seem to lie in the actual freezing. The cells are harvested in the log phase, suspended in a glycerol freezing mixture at five-fold concentration then left overnight (well insulated) in a -70C freeze
Can anybody suggest alternative procedures? The alternatives facing me at present are constructing a "freezing box" which will cool at the correct rate in a -70C freezer (feasible) or persuading the department to buy a commercial cell freezer (less f

Sean Shaw
Microbiology, Reading University
skrshaw@sksscsc1.rdg.ac.uk
Newsgroups: bionet.molbio.methds-reagnts
From: dordie@radium.uio.no (Dordi Anne Aas-Eng)
Subject: T Cells and transfection
Organization: University of Oslo
Date: Wed, 26 Aug 1992 13:15:15 GMT


Do anyone have a efficient method for transient transfection of T-cell
-lines? I am especially interested in methods for transfection of 
Molt  cells, but any method will be of interest. I have tried DEAE-dextran
transfection and electrophoration without great success. 

Please contact me on my e-mail address.

Anne Aas-Eng

   |------------------------------------------------------------------|
   |  Dordi Anne Aas-Eng               dordie@radium.uio.no           |
   |  The Norwegian Radium Hospital                                   |
   |  Dept. of Immunology              Tlf: +47-2-50 60 50 ext. 9744  |
   |  Montebello                       Fax: +47-2-50 07 30            |
   |  N-0310 Oslo                                                     |
   |  NORWAY                                                          |
   |------------------------------------------------------------------|
From: sbhattac@mrc-crc.ac.uk (Dr. Shoumo Bhattacharya B1 061)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Designing oligos for cDNA cloning
Date: 2 Sep 92 12:53:14 GMT
Distribution: bionet

Hi all
Can anyone tell me what programs are available for the designing
of degenerate probes for cDNA cloning.
Many thanks for your help.

Shoumo

Dr. Shoumo Bhattacharya				
MRC Molecular Medicine Group
Royal Postgraduate Medical School
Hammersmith Hospital, DuCane Road London W12 0NN

Newsgroups: bionet.molbio.methds-reagnts
From: kim@m44.unm.edu
Subject: Re: RNAase protection assay...
Date: Wed, 26 Aug 92 16:16:21 GMT
Organization: UNM Cancer Center, Albuquerque, NM
Distribution: bionet
Reply-To: kim@m44.unm.edu

In article <9208261249.AA11697@rac2.wam.umd.edu>, desai@WAM.UMD.EDU (Dinakar) writes:
>
>Hi Molecular Biologists:
>
>Friend of mine is trying to set up RNAse protection assay. I read in previous
>post that there is method to conduct assay directly from GIT. If anyone
>has the reference, I would appreciate receiving the lead to the reference.
>
>Thanks in advance.


See BioTechniques  12(5):736-739.  David Gillespie, one of the authors, has a
kit out that does this, he claims.  I posted a question about this method some
weeks ago with only one reply that it is being tried out by the correspondent's
lab.  If anyone else is doing this, I'd really like to know how it works out. 
I have not had any luck with my own homebrew of this method, but I have had a
number of problems related to my own clumsiness.

Daniel Kim
From: camlau@ubvmsb.cc.buffalo.edu (Joseph T.Y. Lau)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Oligosaccharides
Date: 3 Sep 92 13:02:00 GMT
Distribution: bionet
Organization: University at Buffalo
News-Software: VAX/VMS VNEWS 1.41

In article <9209022155.AA22122@genbank.bio.net>, TY9283@UACSC2.ALBANY.EDU (Jen-Wei) writes...
>Hi,
> 
>   Does anyone know the phone or FAX # for this company, Chembiomed
>Synsorb?
> 
>   Are there anyother companies produce the following compounds?
> 
>      H type-1 oligosaccharide
>      H type-2 oligosaccharide
>      Lacto-N-biose I
>      N-acetyl-2'-O-methyllactosamine
> 
Don't know much about Chembiomed Synsorb.  However, you may want to try

Oxford Glycosystems: (800) 722-2579   
            or
BioCarb Chemicals: (800) 645-6264

Both companies specialize in glycans and glycan related proteins.
Good luck,  J.T.Y. Lau  camlau@ubvms.cc.buffalo.edu
From: gbaa02@udcf.gla.ac.uk (Juliandow)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Drosophila balancer TM3, TM6
Date: 28 Aug 92 16:56:12 GMT
Followup-To: bionet.molbio.methds-reagnts
Organization: GlasgowUni

Can any Drosophila gurus out there help me? I'd like to know the lethal
phases of the TM3 and TM6 balancer chromosomes in homozygotes. Slightly to
my suprise, using the "red book" gave loads of genetic info, but not this
bit.

 - Julian Dow