HDTenEightyP/NetNews huggingface dataset:
From: don@helix.nih.gov (donald seto)
Subject: Re: dye-term removal after cycle seq rxns
Organization: National Institutes of Health, Bethesda
Distribution: bionet
Date: Fri, 11 Sep 1992 12:57:25 GMT

>Yes, I have been doing what you have desribed above, using eppies.  I use
>about 300 ul (not more) of the G-50 gel in a 500 ul eppie that has been 
>I hope there are other better procedures out there.  
>Raj Shankarappa.
>Univ of Pittsburgh
> 
this is for cleaning up fluorescent dye primers for automated
sequencing???
From: bsh@MED.PITT.EDU (Basavaraju Shankarappa)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Subtracted mRNA probes for bacteria?
Date: 10 Sep 92 14:22:41 GMT
Distribution: bionet

> I have been wondering whether it is possible to apply the technique
> of making subtracted cDNA probes from bacterial RNA. I know this is
> pretty routine in the eucaryotic world, but making bacterial cDNA is not.
> What I would like to do is identify whether there are any genes being 
> expressed under one environmental condition which are not expressed under
> another different condition. I don't want to use genetic approaches but
> would like to directly probe southern blots and/or libraries. So is it
> possible to prepare cDNA probes from bacterial mRNA??? (I guess I should
> say RNA since you can't separate the mRNA from the stable RNA). They don't
> need to be full length probes. 
> 
> Would random priming using RT on RNA do the trick? Has this been done before?
> 
> Any and all suggestions welcome.
> 
> 	---------------------------------------------------
> 	 Michael Benedik
> 	 Department of Biochemical and Biophysical Sciences
> 	 University of Houston
> 	 INTERNET: Benedik@UH.EDU	BITNET: Benedik@UHOU

Few crazy ideas:
Since the stable (ribosomal RNA?) is fairly conserved, will it be possible to
make several oligos corresponding to the entire length of the rRNA sequence,
preferably bound on a solid phase like magnetic beads.  Will this enable   
quantitative removal of the rRNA to enable preparation of representative
populations of mRNA.  

It has been reported that ssDNA like primers are resistant to UV inactivation
I guess it is because of their ss nature although it could also be due to 
their relatively small size.  If it is because of their ss nature, is it
possible that RNAs are also similarly resistant.  You may know about this 
better.  If the RNAs are resistant, then you can do an hybrization identical
to the above and then go with UV to inactivate the hybridized RNAs.  If the
above scenario is true then the dsRNAs will form UV mediated dimers which
will not support the subsequent RT reaction.  
Raj Shankarapppa
Univ of Pittsburhg
bsh@med.pitt.edu

Newsgroups: bionet.molbio.methds-reagnts
From: jac209@huxley.anu.edu.au (Juleen A Cavanaugh)
Subject: Re: Chromosomes on disk(ette)?
Organization: Australian National University
Date: Tue, 15 Sep 92 12:20:01 GMT
From: chai_z@wehi.edu.au
Newsgroups: bionet.molbio.methds-reagnts
Subject: Difficulties: M13 cloning
Date: 7 Sep 92 06:10:12 GMT
Organization: Walter & Eliza Hall Institute


Hi.

Anyone could give me ideas about the solving the problems in M13 cloning?

I use lambda primers to PCR the insert from gt10 clone and try to clone 
the PCR product into M13 for sequencing (about 2k). I used to do the same
for some smaller DNAs successfully, but not now. I seems that something 
inhibited the M13. I tried many times with blunt-end and EcoRI site M13
vectors, got only the E.coli cells lawn. The transformation control worked, 
giving a few blue colonies. So, something wrong with the ligation, I meant
something in the reation might inhibit the M13, both wild one, if any, and 
recombinants. What's that?

All the information are appreciated.

Chai_z
From: wmelchior@NTBTOX.NCTR.FDA.GOV ("Bill Melchior, NCTR/FDA")
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Quantitative Autoradiography???
Date: 9 Sep 92 16:49:39 GMT
Reply-To: wmelchior@NTDOC.NCTR.FDA.GOV
Distribution: bionet

>I saw a discussion somewhere recently about the pitfalls of
>using autorads of blots, etc., to estimate quantities of
>things, eg message levels.

I'm not familiar with this exactly, but a related topic is using film to
quantitate fluorescence, as in gel electrophoresis.  D.E. Pulleybank et al
(Nucleic Acids Res., 4#5, 1409-1418 (1977)) discuss the problems.

One of the problems common to both types of measurement is that a film's
response to radiation is linear over only a restricted range, probably
not greater than two orders of magnitude.  
________________________________________________________________________________
The opinions stated are mine, not those of NCTR or its sponsoring organizations.

Bill Melchior                                ||   "You have lawyers the way
National Center for Toxicological Research   ||    other people have mice."
Jefferson, AR  72079                         ||
(501) 543-7206                               ||   -Kenneth Duncan, English
                                             ||    Health & Safety Executive,
WMELCHIOR@NTDOC.NCTR.FDA.GOV                 ||    to US regulators
Organization: Queen's University at Kingston
Date: Friday, 4 Sep 1992 09:20:15 EDT
From: <BACKD@QUCDN.QueensU.CA>
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Luciferase activity by scintillation counting?

Steve,

     I strongly recommend getting a luminometer to do luciferase
assays, particularly if you plan to assay many samples. You can get
a simple luminometer such as the LKB 1250 for < $5000. We used a
scintillation counter in our early assays. It was acceptable, but
a little awkward to use. Please consult the following reference:

Nguyen, V.T., Morange, M. & Bensaude, O. (1988) Firefly luciferase
luminescence assays using scintillation counters for quantitation
in transfected mammalian cells. Anal.Biochem. 171, 404-408

     In the traditional luciferase assay light is generated in a
pulse lasting several hundred milliseconds. Accurate measurements
required that injection be done in the luminometer and the area
under the pulse peak integrated for assay results. K.V. Wood
developed a method that converted the assay from a "flash" to a
"glow" assay (see below). The light production is very stable and
decays with a half time of 5 minutes. This allows measurement of
light intensity in a luminometer very similar to spectrophotometric
measurements. Alternatively, counts per unit time in a
scintillation counter will give similar results. This makes for a
very "gentlemanly" assay. We calculate it costs about $0.04 to $0.1
per assay.

Luciferase "Glow" assay:

5X Cell lysate buffer:

     -125mM Tris-PO4, pH 7.8
     - 10mM CDTA
     - 10mM DTT
     - 50% glycerol
     -  5% Triton X-100

Luciferase assay buffer:

     -20mM Tricine, pH 7.8
     - 1.07mM (MgCO3)4 Mg(OH)2 . 5 H2O
     - 2.67mM MgSO4
     - 0.1mM EDTA
     -33.3mM DTT
     -270Um Coenzyme A, lithium salt
     -470uM Luciferin
     -530uM ATP

Assay:

-Rinse cells 2X with PBS
-Add a minimal volume of 1X lysis buffer, ie 250ul per 60mm dish,
incubate 10 minutes at room temperature.
-scrape cells into a microcentrifuge tube, spin 10 sec in microfuge
-decant supernatant and use for assay. Luciferase is stable for
extended periods when stored at -70C.

-Mix 20ul of extract with 100ul of assay mixture. Mix then place in
luminometer or scintillation counter.

-Use 1X lysate buffer containing 1mg/ml BSA to dilute samples.


Don Back
Department of Biochemistry
Queen's University
Kingston, Ontario
From: coyne@uctvax.uct.ac.za
Newsgroups: bionet.molbio.methds-reagnts
Subject: non-specific cleavage of X-gal
Date: 9 Sep 92 11:06:53 +0200
Organization: University of Cape Town

In article <wakarchu.1.0@biologysx.lan.nrc.ca>, wakarchu@biologysx.lan.nrc.ca (Dr. Warren Wakarchuk) writes:
> In article <1992Sep1.152134.202619@uctvax.uct.ac.za> coyne@uctvax.uct.ac.za writes:
>>From: coyne@uctvax.uct.ac.za
>>Subject: X-GAL CLEAVAGE BY V. CHOLERAE
>>Date: 1 Sep 92 15:21:33 +0200
> 
>>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
>> 
> Your problem is likely that another glycosidase like Beta-glucosidase is 
> cleaving the X-gal.  Most gram negative strains have such enzymes and the 
> specificity is rather "loose".  E. coli Beta-galactosidase does cleave other 
> glucosides as well.  You might have to look for the enzyme itself, not its 
> activity.
> 
> Warren Wakarchuk
> IBS NRCC
> wakarchu@biologysx.lan.nrc.ca

First let me thank Warren for his response to my last posting.  Warren's reply
has prompted the following question:  

If X-gal is such a sensitive substrate for detecting beta-galactosidase
activity, and if most gram negative bacteria possess other glycosidases which
are capable of cleaving X-gal, is it ever possible to get truly "white" 
colonies where lacZ is NOT expressed from an upstream promoter (as in operon
fusions) in a lac- gram negative strain grown on media containing X-gal?  The
answer to this question is important to our current research as we are
attempting to generate lacZ operon fusions using Tn5 in order to identify 
chromosomal genes which respond to specific stresses.

I would be most grateful for any information or suggestions regarding this
problem.

Vernon Coyne 
University of Cape Town
From: sbhattac@crc.ac.uk (Dr. Shoumo Bhattacharya B1 061)
Newsgroups: bionet.molbio.methds-reagnts
Subject: DNA methylation
Date: 9 Sep 92 15:02:41 GMT
Distribution: bionet
Original-To: methods@uk.ac.daresbury

Hi 

Does anyone know of methods to demethylate methylated genes
in tissue culture cells, so that they are accessible to 
trans-acting factors?  I thought of growing them in 
azacytidine, has anyone got a successful protocol for this?

Many thanks.

Shoumo

Dr. Shoumo Bhattacharya				
MRC Molecular Medicine Group
Royal Postgraduate Medical School
Hammersmith Hospital, DuCane Road London W12 0NN
From: MNHVZ082@SIVM.BITNET (Kevin de Queiroz)
Newsgroups: bionet.molbio.evolution
Subject: unsubscribe
Date: 14 Sep 92 21:13:07 GMT
Distribution: bionet

Please unsubscribe me.  Thanks.
From: tabitha@vms.huji.ac.il
Newsgroups: bionet.molbio.evolution
Subject: repetitive DNA
Date: 9 Sep 92 19:23:28 GMT
Distribution: world
Organization: The Hebrew University of Jerusalem

I am a postgraduate student investigating the possible functions of
highly repetitive and especially middle repetitive DNA.  I am persuaded
by the observation that many familieis of middle repetitive DNA are
interspersed with protein coding genes and transcribed.  This may
indicate that they have a regulatory function in gene expression.  Can anybody
help me with references?  These sequences have evolved too slowly -
at about 0.2% per million years - to be junk.  Jeffreys estimated
in the 1982 book Genome Evolution, that if middle repetitive DNA
were junk, it would have evolved at 2% per year.
thanks for any help you can give.
tabitha hofland
tabitha@vms.huji.ac.il
Newsgroups: bionet.molbio.evolution
From: excoffie@sc2a.unige.ch
Subject: Analysis of Molecular Variance : A Windows 3.0 program
Organization: University of Geneva, Switzerland
Date: Tue, 8 Sep 1992 13:15:00 GMT


  This is an annoucement for the public release of a WINDOWS 3.x based program

to analyze the GENETIC STRUCTURE OF POPULATIONS using MOLECULAR INFORMATION.


  This program (WINAMOVA ver 1.04) is based on a paper by Excoffier, Smouse and 

Quattro (1992), and computes variance components for 2 levels of population 

structure as well as their associated modified F-statistics (called here Phi-

statistics).


  This version can handle a maximum of 255 molecular haplotypes, 100 populations

and 100 groups of populations. It requires haplotype frequency information for

each population, as well as a matrix of inter-haplotypic squared (euclidian) 

distances. 


  Conventional multi-allelic F-statistics may also be computed.


  The program includes a non-parametrical (permutational) test for the 

significance of both variance components and Phi-statistics.


  It can be uploaded by anonymous FTP from    : ACASUN1.UNIGE.CH

(username : anonymous) in the directory       :  pub/amova

under the form of an archived file called WINAMOVA.ZIP, including 

executable versions for Windows 3.0 and Windows 3.1, plus example files.


           The program is FREEWARE and can be distributed freely in its 
           original form, but I would appreciate receiving an E-mail from
           interested users, as well as comments, suggestions, and bug
           reports.

(Windows 3.x is a product from Microsoft Corporation)


  Reference cited :
  =================

Excoffier, L, Smouse, PE, and Quattro, JM (1992) Analysis of molecular variance
      inferred from metric distances among DNA haplotypes: Application to human
      mitochondrial DNA restriction data. Genetics 131:479-491.


*******************************************************************************
*                                                                             *
*  Laurent Excoffier                					      *	
*  Genetics and Biometry Laboratory           Tel    : 41-22-343.69.30        *
*  Dept. of Anthropology and Ecology 					      *	
*  University of Geneva                       Fax    : 41-22-300.03.51        *
*  12, rue Gustave Revilliod                  				      *	
*  1227 Carouge-Geneva, Switzerland           E-mail : excoffie@sc2a.unige.ch *
*									      *	
*******************************************************************************
From: NSTEWART@VTVM1.CC.VT.EDU
Newsgroups: bionet.molbio.evolution
Subject: Somatic mutation rates
Date: 12 Sep 92 19:00:40 GMT
Distribution: bionet

Does anyone know of any estimations of somatic mutation rates
for any plant?  References?

Thanks,

Neal
From: GenBank-Updates@genbank.bio.net
Newsgroups: bionet.molbio.genbank.updates
Subject: C. elegans cDNA clone cm11f1 encoding Intermediate filament protein
Date: 10 Sep 92 10:15:26 GMT
Distribution: bionet
Checksum: 27684     2

LOCUS       CEL11F1       336 bp ds-DNA             INV       10-SEP-1992
DEFINITION  C. elegans cDNA clone cm11f1 encoding Intermediate filament protein
            A homologous peptide; 5' end, single read
ACCESSION   Z14375
KEYWORDS    expressed sequence tag.
SOURCE      Caenorhabditis elegans DNA.
  ORGANISM  Caenorhabditis elegans
            Eukaryota; Animalia; Eumetazoa; Nematoda; Secernentea; Rhabditida;
            Rhabditina; Rhabditoidea; Rhabditidae.
REFERENCE   1  (bases 1 to 336)
  AUTHORS   Waterston,R., Martin,C., Craxton,M., Huynh,C., Coulson,A.,
            Hillier,L., Durbin,R.K., Green,P., Shownkeen,R., Halloran,N.,
            Hawkins,T., Wilson,R., Berks,M., Du,Z., Thomas,K., Thierry-Mieg,J.
            and Sulston,J.
  TITLE     A survey of expressed genes in Caenorhabditis elegans
  JOURNAL   Nature Genet. 1, 114-123 (1992)
  STANDARD  full automatic
COMMENT     *source: stage=varied; *source: tissue=whole animal; *source:
            clone_library=Chris Martin sorted cDNA library; *source: sex=
            Hermaphrodite;
            Submitted (16-AUG-1992) to the EMBL Data Library by: Nematode
            Sequencing Project, MRC Laboratory of Molecular Biology, Cambridge
            CB2 2QH, England and Department of Genetics, Washington University,
            St. Louis, MO 63110, USA. E-mail: jes@mrc-lmba.cambridge.ac.uk or
            rw@nematode.wustl.edu
            From EMBL    entry CE11F1;  dated 20-AUG-1992.
BASE COUNT      114 a     67 c     69 g     77 t      9 others
ORIGIN
        1 aaaatgtctg taaacgttcg nccggtgaag cgatttgttg aagaacagaa aaagaaaaat
       61 ccggaaatta aattatctga aaaagccaca caactcctga ctgatatgcc gnaaatggnc
      121 agccctgaga aactgttcaa tctggnngac aaaattgcaa cggaaatggg tagcgatcga
      181 nttttcgaag aatttctcca agattacgca tttttgagaa atgctgttga agaatacttg
      241 gatgganagc caacatcctc ggatactatc tcgaaaatcg cgcgtttacg ccgnnataca
      301 cgacttaccc actatggaac tgccctaatg cgaaaa
//
From: GenBank-Updates@genbank.bio.net
Newsgroups: bionet.molbio.genbank.updates
Subject: Homo sapiens PCR primer binding region and tandem repeat sequence.
Date: 5 Sep 92 10:02:01 GMT
Distribution: bionet
Checksum: 04329     2

LOCUS       HUMRPT44A     275 bp ds-DNA             PRI       05-SEP-1992
DEFINITION  Homo sapiens PCR primer binding region and tandem repeat sequence.
ACCESSION   L02084
KEYWORDS    PCR primer; tandem repeat.
SOURCE      Homo sapiens Blood DNA.
  ORGANISM  Homo sapiens
            Eukaryota; Animalia; Chordata; Vertebrata; Mammalia; Theria;
            Eutheria; Primates; Haplorhini; Catarrhini; Hominidae.
REFERENCE   1  (bases 1 to 275)
  AUTHORS   Weber,J.L. and May,P.E.
  TITLE     Abundant class of Human DNA polymorphisms which can be typed using
            the polymerase chain reaction
  JOURNAL   Am. J. Hum. Genet. 44, 388-396 (1989)
  STANDARD  full automatic
COMMENT     Only one strand sequenced.
FEATURES             Location/Qualifiers
     primer_bind     27..46
                     /standard_name="PCR primer"
                     /evidence=EXPERIMENTAL
     repeat_region   86..115
                     /rpt_family="(CA)n"
                     /rpt_type=tandem
                     /evidence=EXPERIMENTAL
                     /citation=[1]
     primer_bind     complement(131..150)
                     /standard_name="PCR primer"
                     /evidence=EXPERIMENTAL
BASE COUNT       68 a     67 c     55 g     85 t
ORIGIN
        1 gatcttctag tcttgttcat ttacatatct gttcggtcaa tcaggctatt ttatttccac
       61 tgctaaggta cgtatgtgtg tggatacaca cacacacaca cacacacaca cacactcctt
      121 agaccactta ttcccaactt cctccatgag gtgaatgatg gaacgaactg ctctcatttc
      181 tgtccattag gagggtggat ttcccttcac cactgttcct agtagggggg ctataatgga
      241 gtaaggtagg taagcaccat cttgtgctgg cattt
//
From: GenBank-Updates@genbank.bio.net
Newsgroups: bionet.molbio.genbank.updates
Subject: Human immunodeficiency virus type 1 (TAN6-2) V3 neutralizing
Date: 5 Sep 92 10:01:06 GMT
Distribution: bionet
Checksum: 18703     2

LOCUS       HIVTAN62X     275 bp ss-RNA             VRL       05-SEP-1992
DEFINITION  Human immunodeficiency virus type 1 (TAN6-2) V3 neutralizing
            domain.
ACCESSION   L01330
KEYWORDS    envelope protein; neutralizing domain; variable region.
SOURCE      Human immunodeficiency virus type 1 RNA.
  ORGANISM  Human immunodeficiency virus type 1
            Viridae; ss-RNA enveloped viruses; Positive strand RNA virus;
            Retroviridae; Lentivirinae.
REFERENCE   1  (bases 1 to 275)
  AUTHORS   Zwart,G., Wolfs,T.F., Bookelman,R., Hartman,S., Bakker,M.,
            Boucher,C.A., Kuiken,C. and Goudsmit,J.
  TITLE     Greater diversity of the V3 neutralization domain of HIV-1 in
            Tanzania than in the Netherlands; serological and genetic analysis
  JOURNAL   Unpublished (1992)
  STANDARD  full staff_entry
FEATURES             Location/Qualifiers
     misc_feature    1..275
                     /note="V3 neutralization domain"
                     /note="putative"
BASE COUNT      121 a     46 c     44 g     64 t
ORIGIN
        1 agataataat tagatctgaa aatctcacaa ataatgctaa aactataata gtacatctta
       61 atgagtcggt agaaattaat tgcacaaggc cctacaacaa tacaagacaa ggtgtacata
      121 caggaccagg gcaggcactc tttacagaaa aaatagtagg agatataaga caagcacatt
      181 gtaacattag tggaagtagc tgggataaaa ctttacaaca agtagctaac aaattaagag
      241 atcttcttaa caaaacaaca ataatttctc aacca
//
From: GenBank-Updates@genbank.bio.net
Newsgroups: bionet.molbio.genbank.updates
Subject: H.sapiens VH6 mRNA for IG heavy chain V region (A1)
Date: 10 Sep 92 10:33:36 GMT
Distribution: bionet
Checksum: 60634     2

LOCUS       HUMVH6IGA1    372 bp ss-RNA             PRI       10-SEP-1992
DEFINITION  H.sapiens VH6 mRNA for IG heavy chain V region (A1)
ACCESSION   X67890
KEYWORDS    immunoglobulin heavy chain subgroup VH-VI.
SOURCE      Homo sapiens RNA.
  ORGANISM  Homo sapiens
            Eukaryota; Animalia; Chordata; Vertebrata; Mammalia; Theria;
            Eutheria; Primates; Haplorhini; Catarrhini; Hominidae.
REFERENCE   1  (bases 1 to 372)
  AUTHORS   Van Es,J.H., Gmelig-Meyling,F.H. and Logtenberg,T.
  TITLE     High frequency of somatically mutated Igm molecules in the human
            adult blood B cell repertoire
  JOURNAL   Eur. J. Immunol. (1992) In press
  STANDARD  full automatic
COMMENT     *source: cell_type=B cell; *source: clone_library=PCR amplified
            VH6; *source: clone=VH6 A1 through A12; *source: isolate=adult
            blood;
            From EMBL    entry HSVH6IGA1;  dated 18-AUG-1992.
FEATURES             Location/Qualifiers
     CDS             <1..>372
                     /product="immunoglobulin VH6"
                     /codon_start=1
                     /translation="QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPS
                     RGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARD
                     DDSKERNAFDIWGQGTMVTVSS"
BASE COUNT       99 a     94 c     97 g     82 t
ORIGIN
        1 caggtacagc tgcagcagtc aggtccagga ctggtgaagc cctcgcagac cctctcactc
       61 acctgtgcca tctccgggga cagtgtctct agcaacagtg ctgcttggaa ctggatcagg
      121 cagtccccat cgagaggcct tgagtggctg ggaaggacat actacaggtc caagtggtat
      181 aatgattatg cagtatctgt gaaaagtcga ataaccatca acccagacac atccaagaac
      241 cagttctccc tgcagctgaa ctctgtgact cccgaggaca cggctgtgta ttactgtgca
      301 agagatgatg atagtaagga aagaaatgct tttgatatct ggggccaagg gacaatggtc
      361 accgtctctt ca
//