HDTenEightyP/NetNews huggingface dataset:
From: eesnyder@boulder.colorado.edu (Eric E. Snyder)
Newsgroups: bionet.biology.computational
Subject: A Wish-List for Gene Identification Programs
Date: 28 Aug 92 20:46:11 GMT
Distribution: bionet
Organization: University of Colorado, Boulder
Originator: biocomp@lerkim.umiacs.umd.edu

I have been working on a program that identifies coding sequences
from genomic DNA.  I have also been studying the performance of 
a number of the more recent programs/papers on the topic.  I have 
been struck by the singular lack of consistency in the way the 
performance of these systems is evaluated.  While some of this 
inconsistency is due to differences in the limitations or intent 
of the different programs, I think it would be helpful to every-
one to develop a set of standard performance measures by which to
judge the success of each new method.  

I would like to see the following data:

Results in terms of complete sequences:

  Exons (in)correctly predicted (both donor and acceptor sites correct)
  Exons partially predicted (one donor or acceptor site correct)
  Exons partially predicted (prediction overlaps actual exon but 
                             boundaries incorrect)

  Number of exons (of all types) for which the reading frame is (in)correctly 
  predicted

Results in terms of nucleotides:

  Nucleotides (in)correctly predicted as exon  
  Correlation coefficient for exon prediction

Results in terms of splice sites:

  Correlation coefficient for splice site predictions 

Results in terms of assembled genes:

  Number of amino acids in predicted protein (in)correctly predicted



I find that most papers on the subject will analyze their data in terms 
of one or two of these catagories.  Often, it is difficult to get a 
feeling for rates of false positives and never is possible to calculate
the correlation coefficients mentioned above from the raw data.  While
I appreciate one must work within space limitations but there are so many
programs of this type appearing in the literature, it is important to 
be able to attempt some objective comparison.  

So, since I too am working on this problem, I would like to compile 
a wish-list of statistics that bionet readers would like to see.  
What critical bit of information have you found lacking in the analysis
of GeneID, GeneModeler, GRAIL, NETGENE, SORFIND, or whatever program
you have been using.  I will compile a list of such stats and post if 
there is sufficient interest.

Thanks,

Eric E. Snyder                            
Department of MCD Biology              ...making feet for childrens' shoes.
University of Colorado, Boulder   
Boulder, Colorado 80309-0347
From: TJTRUST@UVVM.UVIC.CA
Newsgroups: bionet.journals.contents
Subject: (none)
Date: 26 Aug 92 00:31:42 GMT
Distribution: bionet

unsubscribe bio-journals
From: I.Bowes@uk.ac.southampton ("I.Bowes")
Newsgroups: bionet.molbio.bio-matrix
Subject: join
Date: 26 Aug 92 11:25:18 GMT
Distribution: bionet
Original-To: biomatrx@uk.ac.daresbury
X-Mailer: ELM [version 2.3 PL11]

join biomatrx "Ian Bowes"
Newsgroups: bionet.molbio.methds-reagnts
From: madisa@csuvax1.csu.murdoch.edu.au (Ontiretse Madisa Mine)
Subject: PCR primers containing Inosine?
Keywords: PCR,Inosine,primers
Organization: Murdoch University
Date:  3 Sep 92 03:05:27 GMT

I have been carrying out PCR with primers containing inosine.
For the first 4 weeks there were no problems, but now, no bands
are appearing, despite using low stringency conditions.

My primers are stored at 4 degrees C.  Does anyone have any ideas as to the
cause of my problem?  Is 4 deg C the optimal storage temperature?  How long
can primers remain stable at 4 degrees?

Madisa             email madisa@murdoch.edu.au
From: MBJMR@UK.AC.DARESBURY.DLVH
Newsgroups: bionet.molbio.methds-reagnts
Subject: re* re-use of hyb solutions
Date: 3 Sep 92 14:30:00 GMT
Distribution: bionet
Original-To: METHODS@UK.AC.DARESBURY

a recent item <9282175218.MIN-LVICa26691.bionet-news@uk.ac.daresbury
 
>Subject: URGENT: re-use of hyb solutions
>Bonjour,
>
>worked in a lab once where some people froze their used hybridization solution
>(containing 32P-labelled probe) and, if the probe was still hot enough,
>re-used it for subsequent hybridizations.  They maintained that it worked.
>Frankly, I never tried it, so...
>
>Does anyone out there have experience with re-using frozen hybridization
>solutions containing digoxigenin-labelled oligos?  Does the solution need
>to be boiled before use?  Is getting the salmon sperm DNA into "solution"
>a problem?  Is probe concentration a problem?  Answers to these questions
>and related-experiences greatly appreciated.
>
>Merci beaucoup.
>
>Malcolm
 
I have used digoxigenin-tailed oligonucleotides to probe Southerns.
In my experience, tailed oligos can be stored in hybridization solution at
-20 C and reused at least 5 times.  I just prewarm them to the hybridization
temp while I'm prehybridizing and then replace prehyb. fluid.  I've never had
any problems getting the blocking agents back into solution.
Of course, if you have labelled your oligo by random priming, then the
two strands need to be disociated by heating before use.
Probe concentration is generally not a problem for 4-5 repeat probings, but
you may want to test the amount of probe present after this before using it
again.
 
I hope this helps.
 
Janet
 
mbjmr@dlvh.dl.ac.uk
Newsgroups: bionet.molbio.methds-reagnts
From: twomey@helix.nih.gov
Subject: freezers
Organization: National Cancer Institute
Date: Wed, 2 Sep 1992 01:37:29 GMT

Hello,

In a former lifetime, I vaguely remember seeing small freezers advertised for
sale as "personal" restriction enzyme freezers.  They were small enough to fit
on the benchtop and were specifically designed for storage of molecular biology
enzymes.  (I'm refering to actual mechanical freezers, not the little cold
blocks for temporary cooling).  Now that I want one, I can't find a source
anywhere.  Does anyone know where I can find such a thing?

Thanks,
Patrick Twomey
twomey@helix.nih.gov
From: horton@molbio (Robert Horton)
Newsgroups: bionet.molbio.methds-reagnts
Subject: poly A selection
Date: 26 Aug 92 03:20:39 GMT
Organization: University of Minnesota


	Greetings, denizens of cyberspace.
I have some theoretical/technical questions regarding selection of poly A+
RNA on oligo dT:
	1) I've seen protocols for doing RNA hybridizations (e.g., for RNase
protection assays) in a guanidine solution. This supposedly has two effects;
it speeds up hybridization (however that works) and prevents any nasty RNases
from degrading the RNA prematurely. Could a guanidine-containing buffer be 
used with an oligo dT cellulose column? (I get so nervous when my RNA has to 
sit around at room temperature in water for an hour or so while I run these
columns).
	1a) Can you keep azide (or something) on these columns all the time
to keep bugs from even thinking of growing on them? (And wouldn't they last
forever if nothing ate them?)
	2) Is there a better method nowadays than oligo dT cellulose (e.g., 
magnetic beads, dT columns for HPLC, etc) that I am (hopefully) just unaware
of? (Dare I ask about kits people may like?)B-)) It seems like you could speed
up the process with a non-compressible matrix and a syringe (a poor man's 
HPLC), but I'd hate to re-invent that wheel if somebody already sells 'em...
	3) Or maybe I don't really need poly A+ RNA (I'm trying to label mRNA
for use as a probe on a panel of cloned genes. I've tried making hot cDNA from
total RNA using an oligo dT primer, but this gives a monstrous background).
	Comments and suggestions would be greatly appreciated.
--
Bob Horton            /\ "Crash programs fail because of the theory that
U. of Minnesota, CBS  || with nine women pregnant you get a baby a month" 
1479 Gortner Ave.    /||\   -Werner von Braun.  Disclaimer:"Bob who?"
St. Paul, MN 55108    ^^   horton@molbio.cbs.umn.edu/(612) 624-3790
From: saparici@crc.ac.uk (Dr. S.A.J.R. Aparicio)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: PCR primers containing Inosine?
Keywords: PCR,Inosine,primers
Date: 3 Sep 92 10:51:24 GMT
Reply-To: saparici@crc.ac.uk (Dr. S.A.J.R. Aparicio)
Organization: University of Cambridge, Department of Medicine


We've never experienced instability of Inosine primers although ours
are stored at -20C. Once or twice, we have found that buffers etc stored at
4C, which are in frequent use can become contaminated with fungi/bacteria and
this has stopped reactions from working.

I know this sounds obvious and you've probably checked it out already but I would
make sure that things such as dNTP's and buffer are fresh and working. They are
far more labile than primers.


Sam Aparicio
MRC Molecular Genetics Unit
Cambridge
CB2 2QH
Organization: Queen's University at Kingston
Date: Wednesday, 26 Aug 1992 08:25:34 EDT
From: <BACKD@QUCDN.QueensU.CA>
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: genbank e-mail searching
Distribution: bionet

Send a 1 line help message to each of the following servers to get
information on searches.
Leave the subject line blank
Type HELP on the first line

FASTA ---  SEARCH@GENBANK.BIO.NET
BLAST ---  BLAST@GENBANK.BIO.NET
BLAZE ---  BLAZE@GENBANK.BIO.NET
Don Back
Department of Biochemistry
Queen's University
Kingston, Ontario. CANADA.
From: ray@uk.ac.le ("Dr R. Dalgleish")
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: PBR322 - maximizing yields
Date: 27 Aug 92 15:47:29 GMT
Distribution: bionet
Original-To: bhjelle@edu.unm.carina
Original-Cc: methods@uk.ac.daresbury
X-Mailer: ELM [version 2.3 PL0 (LUT)]

> 
> What are the best tricks for increasing yields of pBR-based
> vectors?
> 
> Brian
> 
> 
The way which I used to amplify pBR-type vectors was this:

Add chloramphenicol to a final concentration of 200 micrograms per ml to
late log phase cells. If you add it too late it won't help. For large size
cultures you can weigh out the powder and just tip it in and shake. Folklore
says not to shake too vigourously. If you are working with smaller culture
then make a 34 mg/ml solution of choramphenicol in 50% ethanol and add the
appropriate amount.

Of course, be sure that your vector does not carry a Cm resistance gene! If
it does then you can amplify using spectinomycin. The amount to use escapes
me at the moment.

Raymond Dalgleish.
Newsgroups: bionet.molbio.methds-reagnts
From: lhale@upei.ca (Larry Hale)
Subject: Electroblotting
Keywords: Southern transfer/acrylamide/ds DNA
Organization: University of Prince Edward Island
Date: Tue, 1 Sep 1992 14:06:24 GMT

Can anyone suggest a place to find a reliable protocol for transfer of
double stranded DNA from SDS-PAGE gels to an appropriate medium (hopefully
nylon membranes)? I have a new unit from EC Apparatus (EC-217), but the 
manuals are not terribly helpful. In particular, I need to know the steps 
required (if any) required to prepare the DNA for probe hybridization from 
such a gel.

Thanks, L.Hale, UPEI, Charlottetown, Canada

Newsgroups: bionet.molbio.methds-reagnts
From: browns@ccu.umanitoba.ca (Stuart Brown)
Subject: Re: cDNA library kits
Organization: University of Manitoba, Winnipeg, Manitoba, Canada
Distribution: bionet
Date: Fri, 4 Sep 1992 15:18:06 GMT

In article <1992Sep3.142153.27303@gserv1.dl.ac.uk> PHILLIPSA@UK.AC.AFRC.LARS (Andy Phillips) writes:
>I have a visiting worker in the lab who has produced some
>PCR products and now needs to screen a library for full-length
>cDNAs. He only has 4 months in the lab; the last time I made a library,
>using a cDNA synthesis kit but then making the rest up as I went
>along, it took me nearly that long to sort everything out. Which
>cDNA library kit would you people out there recommend? We want
>it to work first time (Ha Ha Ha), and preferably to include all
>steps from mRNA to packaged library (so that we can ask for our
>money back if it doesn't work). Please mail me with all your
>experiences of such kits (positive or negative).
>
>Thanks in advance
>
>Andy Phillips
>Long Ashton Research Stn,
>Bristol, UK

You should also check out whether there are commercailly available pre-made
libraries that would suit your purposes (or if one could be borrowed from
another group).  Also there are a couple of companies that will make a 
library for you from your mRNA.  Depends if you have the $$ to make up for
the lack of time - but I think it's a good idea to rely on someone who
specializes in a particular technique rather than re-inventing it for
yourself.

-- 
Stuart M. Brown                             If you can remain cool when all 
U. of Manitoba, Dept. Plant Science         Around you are in panic,
Winnipeg, Manitoba, CANADA
browns@ccu.umanitoba.ca            Then you surely misunderstand the situation
From: LLR8156%USA.decnet@USAV01.GLAXO.COM ("USA::LLR8156")
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re:N2 storage of murine cell lines
Date: 2 Sep 92 20:30:00 GMT
Distribution: bionet


     Nalgene sells a cryo freezing container with a colling rate of
-1 degree C/minute.  To use, you simply fill the inner container with
isopropyl alcohol and store at -70 for a few hours.  It's relatively
inexpensive- about $35, and holds 18 cryovials.  You can also purchase it
through suppliers like Fisher or Research Products International.
     Have you tried freezing down your cell lines with 5-10 % DMSO
instead of glycerol?  For several cell lines I've worked with, I get
better viability with DMSO compared to glycerol.

Best wishes;
Lisa Ross
Glaxo Research Institute, Inc.

Newsgroups: bionet.molbio.methds-reagnts
From: a52041@sakura.kudpc.kyoto-u.ac.jp (Michael CHENG)
Subject: Sf9 Cells
Originator: a52041@sakura.kudpc.kyoto-u.ac.jp
Organization: Data Processing Center, Kyoto University, Kyoto Japan.
Date: Sat, 29 Aug 1992 06:07:01 GMT

  Hi! I was wondering if anyone out there has experience working with Sf9 cells
isolated from the ovary of armyworms.
  I pretty much follow the protocol of Summers and Smith. Using Grace's medium
supplemented with 10% FCS and the specified concentration of Gentamicin.
  However, recently my cells begin to die after a few days of incubation.
Specifically, after reviving the cells from liquid N2 and seeding 2e6 cells in
a 25cm2 flask, the cells are fine and reaches confluency. I would then pass the
cells 1 to 3 or 1 to 4 in about 5 days. But appoximately 2 days after the 1st
passage, the cells would begin to die. They would float, stop dividing, and I
think that I can see holes in them (some bright spots?). Don't think it's
contamination by baculovirus though that's what I'm growing the cells for?
Has anyone experienced this? Any suggestions?
  Are these cells susceptible to mycoplasma contamination? The gentamicin that
I'm using is from BRL and the maker describes as effective against mycoplasma.
  I've also heard that a "new?" medium called Sf900 is better. Any comments?
   How many passages can I achieve with these cells continously
  Right now, I plan on purchasing new cells from INV.
  I would appreciate any comments or suggestions.
  Thanks in advance.

a52041@sakura.kudpc.kyoto-u.ac.jp
Michael Cheng
?
From: desai@WAM.UMD.EDU (Dinakar)
Newsgroups: bionet.molbio.methds-reagnts
Subject: RNAase protection assay...
Date: 26 Aug 92 12:49:43 GMT
Distribution: bionet


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.

Dinakar
BARC-EAST, USDA
From: Klaus.Matthaei@anu.edu.au
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: PCR from few cells.
Date: 26 Aug 92 22:13:54 GMT
Distribution: bionet

>Hello.
>Does anyone out there have a 
>protocol for using the PCR on the DNA from
>a small number of cell; 10-1000 mamalian cells?
>Ideally this will be able to work from tissue culture cells 
>being cloned by limiting dilutions in 96 well
>flat-bottomed tissue culture plates.  Thanks in advance.
>
>Mark Brudnak
>University of Tulsa
>INTERNET: BMA14432@VAX1.UTULSA.EDU

G'Day
Some years ago we developed a PCR method for sexing pre-implantation cattle
embryos during commercial embryo transfer.  This was usually done on the
kitchen table in the farmhouse so the method had to be simple and
foolproof.  If your numbers of cells is not too high (< 500-1000), wash
your cells and resuspend in 10-20  l of water.  Boil 10 minutes and add the
rest of your PCR cocktail  (we used to make a mix containing  everthing
including the taq and freeze in Liquid nitrogen) to say 50  l, add oil and
cycle.  We are able to accurately sex a single cell this way (~1000 target
molecules since it was a repeat) which we obtain by biopsy of the embryo at
the blastocust or morula stage. 

Hope this helps.

Cheers, Klaus.
----------------------------------------------------------------------------
Klaus Matthaei
Gene Targeting
The John Curtin School of Medical Research
The Australian National University
E-mail: Klaus.Matthaei@anu.edu.au

"Don't try and fix it if it ain't broke".
----------------------------------------------------------------------------
From: axa12@po.CWRU.Edu (Ashok Aiyar)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Partial digestions with ApaL1 - help!! - THANK YOU
Date: 2 Sep 1992 14:27:30 GMT
Organization: Case Western Reserve University, Cleveland, OH (USA)
Reply-To: axa12@po.CWRU.Edu (Ashok Aiyar)
NNTP-Posting-Host: thor.ins.cwru.edu



I thank all those who replied.  Several people suggested adding
in EtBr at very low levels.  Others recommended using sub-optimal
conditions such as adding high salt or a sub-optimal buffer.

I changed the buffer in which I *diluted* the enzyme, and used
the Sequenase dilution buffer instead.  This seems to be working.

My original buffer had 50% glycerol, 100mM NaCl, 10 mM BME, 0.1mM
EDTA and 20 mM Tris pH 7.5
The Sequenase buffer contains 10 mM Tris pH 7.5, 5 mM DTT, and
0.5 mg/ml BSA

I apologize for not replying individually to those who replied,
but I really *have* to get this cloning done :-)

Regards to all,

Ashok
-- 
                          Ashok Aiyar
   		       axa12@po.cwru.edu
                Visit the IBM-PC Sig on Freenet
From: BMA14432@VAX1.UTULSA.EDU
Newsgroups: bionet.molbio.methds-reagnts
Subject: Tacking down cells
Date: 27 Aug 92 17:27:07 GMT
Distribution: bionet


Hello netters,
I recently read somewhere about a product that will "tack down"
non adherant cell to a dish or plate. Does anyone know of this product. Where 
can I get it or how can I make it? Also, any other information concerning
its ability to do this is also apprecitated.
Mark Brudnak
University of Tulsa
INTERNET: BMA14432@VAX1.UTULSA.EDU
INTERNET:BMA14432@VAX1.UTULSA.EDU
Newsgroups: bionet.molbio.methds-reagnts
From: ca566@cleveland.Freenet.Edu (Michael Holloway)
Subject: Free alpha 33P dATP from NEN
Reply-To: ca566@cleveland.Freenet.Edu (Michael Holloway)
Organization: Case Western Reserve University, Cleveland, OH (USA)
Date: Thu, 27 Aug 92 14:36:31 GMT


There had been some chatter about using 33P in sequencing here last week. 
I bumped into our NEN salesperson yesterday and she asked me to pass on an
invitation to anyone that was interested in trying a free shipment of 33P. 
Just leave a message at her voice mail number and she'll get back to you:
Susan B. Anderson, 800-453-8869, box 111-9171.

-- 
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: PHILLIPSA@UK.AC.AFRC.LARS (Andy Phillips)
Newsgroups: bionet.molbio.methds-reagnts
Subject: cDNA library kits
Date: 3 Sep 92 15:23:00 GMT
Distribution: bionet
Original-To: METHODS@UK.AC.DARESBURY.DLGM

I have a visiting worker in the lab who has produced some
PCR products and now needs to screen a library for full-length
cDNAs. He only has 4 months in the lab; the last time I made a library,
using a cDNA synthesis kit but then making the rest up as I went
along, it took me nearly that long to sort everything out. Which
cDNA library kit would you people out there recommend? We want
it to work first time (Ha Ha Ha), and preferably to include all
steps from mRNA to packaged library (so that we can ask for our
money back if it doesn't work). Please mail me with all your
experiences of such kits (positive or negative).

Thanks in advance

Andy Phillips
Long Ashton Research Stn,
Bristol, UK
From: BMA14432@VAX1.UTULSA.EDU
Newsgroups: bionet.molbio.methds-reagnts
Subject: PCR from few cells.
Date: 26 Aug 92 19:23:01 GMT
Distribution: bionet

Hello.
Does anyone out there have a 
protocol for using the PCR on the DNA from
a small number of cell; 10-1000 mamalian cells?
Ideally this will be able to work from tissue culture cells 
being cloned by limiting dilutions in 96 well
flat-bottomed tissue culture plates.  Thanks in advance.

Mark Brudnak
University of Tulsa
INTERNET: BMA14432@VAX1.UTULSA.EDU
From: TY9283@UACSC2.ALBANY.EDU ("J.W.")
Newsgroups: bionet.molbio.methds-reagnts
Subject: Chembiomed Synsorb
Date: 3 Sep 92 19:40:33 GMT
Distribution: bionet

Hi,

   Does anyone know the number for Chembiomed Synsorb?

   I am trying to locate the following oligosaccharides:

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

  Thanks in advance for any suggestion.
                                                       Jen-Wei
From: pnh@fcs260c2.ncifcrf.gov (Paul N Hengen)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Problem isolating M13 ssDNA
Date: 28 Aug 92 16:51:48 GMT
Distribution: bionet
Organization: Frederick Cancer Research and Development Center

In article <1992Aug28.004702.3463@nrcnet0.nrc.ca> num208ww@MBDS.NRC.CA writes:

>>Usually, I work with dsDNA but on occasion I make ssDNA to
>>sequence troublesome clones.  

*stuff deleted*

>>- Steve Klautky -

>1) In my hands DH5-alpha-F' is a low yield producer of ssDNA, I've had
>much better luck with MV1190 (or even JM101 in the olden days)...

You may have to try a number of F' strains to get the desired ssDNA.
There's a strain from Bethesda Research Labs called DH11S and one
from Statagene called XL1-blue.

>2) Your "troublesome clones" may be res[p]onsible for your decreased
>yields, many clones simply do not produce ssDNA well...

>Dr. Warren Wakarchuk

This is a FAQ for this group. You may find helpful hints for
trouble shooting in the archives. Does anyone keep a FAQ list
for this group?

Paul N. Hengen
National Cancer Institute
Frederick Cancer Research and Development Center
Frederick, Maryland 21702-1201 USA