HDTenEightyP/NetNews huggingface dataset:
From: cytogen@chmeds.ac.nz (Martin Kennedy)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: lambda digest: the pen-ultimate solution ?
Date: 12 Sep 92 09:51:14 GMT
Distribution: bionet

In article <1992Sep11.131419.25990@gserv1.dl.ac.uk>, suter@VAX.MPIZ-KOELN.MPG.dbp.de writes:
> Mike Grosse wrote:
> 
> <Subj:	lambda won't cut
> <
> <Hello netters,
> <
> <A colleague provided us with some lambda clone DNAs "purified" from plate
> <lysates.  They won't cut with EcoR1.  We tried further purification with
> <additional phenol/chloroform extractions.  The uncut DNA looks good on a
> <gel, and the OD's are good.  The inserts should cut out with EcoR1 based on
> <the way the library was made (R1 linkers).  We haven't worked with lambda
> <in quite a while, but I imagine that some of you out there are cutting
> <yours successfully as you read this.
> <
> <What do you suggest?
> <
> <                                Mike Grosse
> <                                McGraw lab
> <                                University of Georgia
> 
> i had EXACTLY the same problem two months ago: same enzyme, same technique,
> same results ! my boss suggested that there is something in the LB/top agarose
> that inhibits digestion, and you won't be able to get rid of it with 
> standard procedures (heavy metal ? polysaccharides ? i don't know). anyway,
> these same compounds may trouble you when you ligate in low melting agarose
> also...
> 
> after many different purifications, all in vain, i purified the DNA
> over a QIAGEN column, and presto: perfect DNA, perfect Eco digest, perfect
> ligation.
> 
> if you don't have these kind of columns, perhaps you should use another
> kind that is used for DNA preparation in your lab, i just followed
> the dna miniprep protocol (perhaps sephadex would also work, altho the principal
> is completely different).
> 
> good luck !
> 
> clemens
> 

A few years back I read a paper (sorry - can't remember where, but possibly
Gene Analysis Techniques)  where someone tried numerous procedures to remove
the unidentified nasty(s) which inhibit cutting of lambda preps.  They claimed
that passing the lambda DNA through a Sepharose 4B (or 6B - again, my memory
fails me) column usually solved the problem. I haven't tried it.  Why not
transform the DNA back into E.coli  (works at low frequency) and make a new
prep (I always find new preps more likely to work than trying to get old ones
to cut)?  The other desparate measure I've used is multiple phenol/CHCl3
extractions (ie 5 or 6), which occasionally works.

Cheers,

Martin
From: bionet@EVOL.BIOLOGY.MCMASTER.CA (bionet)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Chemiluminescent sequencing
Date: 9 Sep 92 12:53:22 GMT
Distribution: bionet

I am interested in anyone that has had experience with
chemiluminescent sequencing.  Particularly the DIG
(digoxigenin) from Boehringer Mannheim.  

Any experiences would be welcome, especially with regard
to how sensitive it is in comparison with S35/P32 sequencing.

Thanks
Brian Golding, Department of Biology
McMaster Univ., Hamilton, Ontario
(brian@evol.biology.mcmaster.ca);
Newsgroups: bionet.molbio.methds-reagnts
From: jing@milton.u.washington.edu (Jing Huang)
Subject: Re: Lambda won't cut
Reply-To: jing@u.washington.edu
Organization: University of Washington, Seattle
Date: Thu, 10 Sep 1992 17:50:27 GMT

What kind of plate did you get the lysate from?  Bacto agar contains 
polysaccharides that inhibit restriction cut.  Using agarose plate 
will solve the problem.

Good luck!

-Jing Huang
jing@u.washington.edu

Newsgroups: bionet.molbio.methds-reagnts
From: bjoernf@ulrik.uio.no (Bj|rn Forbord)
Subject: Lysis of bacteria
Organization: University of Oslo, Norway
Distribution: bionet
Date: Tue, 15 Sep 1992 08:32:01 GMT

We are currently running PCR on samples consisting of a number of different
bacterial strains.  We don't know which bugs are in the samples and are
utilizing a number of primers in this endeavour.  Our main concern is this:
How do we know whether we really are able to lyse any bacterium, regardless
of cell wall composition etc.?
Can anyone out there suggest good lysing techniques that don't interfere with
PCR amplification?
--
Bjoern Forbord
(bjoernf@karius.uio.no)
Odont Inst Physiol Biochem
Dental Faculty
PO Box 1052 Blindern
N-0316 OSLO 3
Tel: +47 2 856029
Fax: +47 2 856025
Secretary: +47 2 854462
Newsgroups: bionet.molbio.methds-reagnts
From: gat44@iastate.edu (Gabriel Trueba)
Subject: Re: PCR with E. Coli
Organization: Iowa State University, Ames, IA
Distribution: bionet
Date: Sat, 5 Sep 1992 23:55:24 GMT

 We normally amplify segments of 4-6 kb using 3 min extensions.

Good luck!
Fri, 11 Sep 1992 12: 05:55 +0100
               Thu, 10 Sep 1992 15: 26:54 +0100
               Thu, 10 Sep 1992 18: 32:46 +0100
From: suter@VAX.MPIZ-KOELN.MPG.dbp.de
Newsgroups: bionet.molbio.methds-reagnts
Subject: lambda digest: the pen-ultimate solution ?
Date: 10 Sep 92 17:32:46 GMT
Distribution: bionet
Content-Identifier: 12
Original-To: methods@uk.ac.daresbury
Conversion: Prohibited

Mike Grosse wrote:

<Subj:	lambda won't cut
<
<Hello netters,
<
<A colleague provided us with some lambda clone DNAs "purified" from plate
<lysates.  They won't cut with EcoR1.  We tried further purification with
<additional phenol/chloroform extractions.  The uncut DNA looks good on a
<gel, and the OD's are good.  The inserts should cut out with EcoR1 based on
<the way the library was made (R1 linkers).  We haven't worked with lambda
<in quite a while, but I imagine that some of you out there are cutting
<yours successfully as you read this.
<
<What do you suggest?
<
<                                Mike Grosse
<                                McGraw lab
<                                University of Georgia

i had EXACTLY the same problem two months ago: same enzyme, same technique,
same results ! my boss suggested that there is something in the LB/top agarose
that inhibits digestion, and you won't be able to get rid of it with 
standard procedures (heavy metal ? polysaccharides ? i don't know). anyway,
these same compounds may trouble you when you ligate in low melting agarose
also...

after many different purifications, all in vain, i purified the DNA
over a QIAGEN column, and presto: perfect DNA, perfect Eco digest, perfect
ligation.

if you don't have these kind of columns, perhaps you should use another
kind that is used for DNA preparation in your lab, i just followed
the dna miniprep protocol (perhaps sephadex would also work, altho the principal
is completely different).

good luck !

clemens
----------------------------------> cut here <----------------------------------
Clemens Suter-Crazzolara, PhD
Max-Planck-Institut fuer Zuechtungsforschung
Abteilung Genetische Grundlagen der Zuechtungsforschung
Carl-von-Linne Weg 10
5000 Koeln 30
Tel. xx49-221-5062.221           fax. xx49-221-5062.21
suter@vax.mpiz-koeln.mpg.dbp.de
------------------------------------------------------------------------------- 
     ! all job offers should include a self-addressed, stamped envelope !
-------------------------------------------------------------------------------
Newsgroups: bionet.molbio.methds-reagnts
From: ALSOBROOK@biomed.med.yale.edu (John Alsobrook)
Subject: Re: Epstein Barr
Organization: Yale University
Date: Tue, 15 Sep 1992 17:22:48 GMT
X-News-Reader: VMS NEWS 1.20

In <1992Sep8.213536.22749@gserv1.dl.ac.uk> sbhattac@crc.ac.uk writes:

> Just a comment.
> 
> Epstein Barr virus is used to 'immortalize' lymphoblastoid cells from 
> humans. Avoid transforming cell lines of laboratory staff as accidental
> self inoculation with your own immortalised cell line will not be a 
> good thing.
> 
> Dr. Shoumo Bhattacharya				
> MRC Molecular Medicine Group
> Royal Postgraduate Medical School
> Hammersmith Hospital, DuCane Road London W12 0NN


We also use EBV for the same purpose; our employee-health department required 
a blood test to demonstrate a positive anti-EBV antibody titer before working
with it. Evidently most folks (at least in the U.S.) have titers from exposures
during childhood/adolescence. 

And of course, Dr. Bhattacharya is absolutley correct!! You don't want any
immortalized cells in your body that are blissfully accepted as 'self' !

****************************************************************************
*                                       | "Giving money and power to the   *
* John Alsobrook                        |    government is like giving     *
* Child Study Center                    |     whiskey and car keys to      *
* Yale University School of Medicine    |  teenage boys." - P.J. O'Rourke  *
*--------------------------------------------------------------------------*
*                       blame me, not my parents!                          * 
*                  bitnet: alsobrook@yalemed.bitnet                        *
*               internet:alsobrook@biomed.med.yale.edu                     *
*--------------------------------------------------------------------------*
...and, of course, nothing I ever say is officially sanctioned by Yale...
From: s882076@yallara.cs.rmit.OZ.AU (Sean Davidson)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Help: Transfection into mammalian cells
Date: 15 Sep 92 12:09:28 GMT

jing@milton.u.washington.edu (Jing Huang) writes:

>Hi, I am trying to co-transfect 2 plasmids into both 3T3 (fibroblasts) 
>and C2 (myoblasts) cells.  I am doing transient transfection and I need
>to be able to control both the ratio of the 2 plasmids and the absolute
>copy number of each plasmid inside a single cell.  The ratio can be 
>achieved by the calcium phosphate method which gets multiple copies of 
>plasmids into a cell, while electroporation usually gets only 1 copy of 
>plasmid into each cell.  Does anyone know how to limit the copies of 
>DNA molecules in the Ca method?  and how to get more than 1 but defined 
>number of plasmids by electroporation?  Thanks! and I will post a summary.

I don't know if this method will be any better, but a rep from Boehringer
recently told me they now sell a kit for transfection which used
liposomes. Sorry I don't know any more but I'm sure they will gladly
tell you about it.
Sean
From: skspoidn@susssys1.reading.ac.uk
Newsgroups: bionet.molbio.methds-reagnts
Subject: re ligation trouble
Date: 9 Sep 92 09:01:35 GMT
Distribution: bionet
Original-To: methods@uk.ac.daresbury

re SphI ligations:

have you tried sacrificing a chicken under a full moon? :)

Seriously, 3 small suggestions, because 
I have difficulty with 3' overhangers as well

1) You don't mention if the alk phos treatment worked. I find that 3'
 overhangs are much harder to dephos than 5' or even blunt.

2) What is the composition of your ligase buffer? We recently changed 
to Promega and were horrified to discover that the buffer as supplied
 contains no condensation agents (usu glycerol or PEG), and that these
 agents should be added by the user if neccessary. You could try upping 
these components to max levels and see if that helps.

3) Without wishing to appear arrogant, condescending or patronising,
 have you ensured that your ratio of insert to vector is appropriate?

Hope this helps,

Mike Poidinger
Dept of Microbiology
University of Reading
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: DNA methylation
From: cytogen@chmeds.ac.nz (Martin Kennedy)
Date: 10 Sep 92 11:08:08 +1300
Distribution: bionet

In article <1992Sep9.150805.11081@gserv1.dl.ac.uk>, sbhattac@crc.ac.uk (Dr. Shoumo Bhattacharya B1 061) writes:
> 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


Shoumo-

The following reference might be helpful:

Dobkin et al (1987)  PFGE of human DNA: 5-azacytidine improves restricition
Nucl. Acids Res. 15, 3183.

Cheers,

Martin
Newsgroups: bionet.molbio.methds-reagnts
From: horton@molbio.cbs.umn.edu (Robert Horton)
Subject: Re: Acidic polyacrylamide gels
Organization: University of Minnesota
X-Newsreader: Tin 1.1 PL5
Distribution: bionet
Date: Mon, 14 Sep 1992 17:37:33 GMT

Pardon my ignorance (I'm one of those "molecular biologists" who practices
biochemistry without a license), but couldn't you just hook the electrodes up
the other way? A basic protein should migrate towards the negative electrode ...
?
--
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
Newsgroups: bionet.molbio.methds-reagnts
From: John.Hardham@bbs.oit.unc.edu (John Hardham)
Subject: Re: ASAP
Organization: Extended Bulletin Board Service
Distribution: bionet
Date: Wed, 9 Sep 1992 19:03:17 GMT

In article <1992Sep9.151940.11625@gserv1.dl.ac.uk> skspoidn@susssys1.reading.ac.uk writes:
>re Arctic Shrimp Alk Phos for dephos treatment:
>Really? Arctic Shrimp?  I mean, how did they find out?
> Is there someone out there who tests different enzymes
> from different sources to see how good they are? 
>(Have you tried the poodle S1 nuclease?) :)....
>
>Oh, I have it! The BIG CHEESES worked this one out,
> and since BIG CHEESES only communicate with 
>BIG CHEESES, I never got to hear of it.
>
>Mike

Yes, there really is an Artic shrimp alk. phos.
I have no idea how they found this out. Nor do I know who would be testing
artic shrimp for alkaline phosphatase activity in the first place.  But
if you check the USB catalog, it is there.  Even more suprising is that
it really works.  Best part is that you can digest and SAP at the
same time.



--
   The opinions expressed are not necessarily those of the University of
     North Carolina at Chapel Hill, the Campus Office for Information
        Technology, or the Experimental Bulletin Board Service.
           internet:  bbs.oit.unc.edu or 152.2.22.80
Newsgroups: bionet.molbio.methds-reagnts
From: bchs1b@Elroy.UH.EDU (Michael Benedik)
Subject: Subtracted mRNA probes for bacteria?
Reply-To: bchs1b@Elroy.UH.EDU
Organization: University of Houston
Date: Thu, 10 Sep 1992 05:08:16 GMT

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
Newsgroups: bionet.molbio.methds-reagnts
From: mfarrall@crc.ac.uk (Martin Farrall)
Subject: Re: lambda won't cut
Reply-To: mfarrall@crc.ac.uk (Martin Farrall)
Organization: MRC Human Genome Mapping Project Resource Centre, Harrow, UK
Distribution: bionet
Date: Thu, 10 Sep 1992 07:25:07 GMT

In article <9209091448.AA23264@server.uga.edu>, mcgraw%gandal.dnet@SERVER.UGA.EDU writes:
> A colleague provided us with some lambda clone DNAs "purified" from plate
> lysates.  They won't cut with EcoR1.  We tried further purification with
> additional phenol/chloroform extractions.  The uncut DNA looks good on a
> gel, and the OD's are good.  The inserts should cut out with EcoR1 based on
> the way the library was made (R1 linkers).
> 
> What do you suggest?



Was the DNA prepared using a PEG precipitation method?  Folk-lore has it that PEG contamination
inhibits various enzymes including restriction enzymes.  I recommend the use of one of the
non-PEG methods for lambda preps e.g. LambdaSorb (Promega), which has yielded a reasonable yield
of cuttable DNA nearly all the time in our hands.

Martin Farrall
mfarrall@rpms.ac.uk

P.S. I forgot to mention that according to Maniatis et al., agar sometimes contains an inhibitor
of restriction enzymes so it is recommended to use agarose as a substitute.
From: wakarchu@biologysx.lan.nrc.ca (Dr. Warren Wakarchuk)
Newsgroups: bionet.molbio.methds-reagnts
Subject: GF/C  filters for ssDNA
Date: 11 Sep 92 16:00:58 GMT
Organization: National Research Council of Canada

Hi Netters.  Enough people have asked me for the reference to the method I 
mentioned in passing a while ago that I thought I'd post it for all to see.  
The method is for the isolation of ssDNA from either M13 or phagemid culture 
supernatents.  It uses GF/C glass fibre filters and Sodium perchlorate. The 
reference is:

   Kristensen et al., Nucleic acids research 15(14): 5507 - 5516, 1987

Cheers,
Warren Wakarchuk              Email:  WAKARCHU@BIOLOGYSX.LAN.NRC.CA
Protein Structure and Design          NUM208WW@MBDS.NRC.CA
IBS-NRCC                        fax:  613-952-9092
Ottawa, Ont.  CANADA
**Any opinions stated here are my own, not NRC's**
From: nwhite@crc.ac.uk (Nick White)
Newsgroups: bionet.molbio.methds-reagnts
Subject: pMX Vector
Date: 7 Sep 92 13:14:30 GMT
Distribution: bionet
Original-To: methods@uk.ac.daresbury


Hello Bionetters,

Has anybody heard of the mammalian COS expression vector pXM ? I want to
use it to transfect some mouse fibroblasts but I desparately need
its restriction map!

If anyone has a copy or knows of this vector's source could you please mail it 
to me.

Thanks in advance!

Adele Murrell
Cambridge University

I'm only borrowing this account - please mail replie to amm@mrc-lmb.cam.ac.uk
From: klopfer@natinst.com (Mike Klopfer)
Newsgroups: bionet.molbio.methds-reagnts
Subject: reconstitutions of proteins in a bilipid membrane
Date: 8 Sep 1992 12:02:39 -0500
Organization: National Instruments, Austin, TX
NNTP-Posting-Host: eagle.natinst.com

From klopfer@cerc.utexas.edu Mon Sep  7 21:38:22 1992
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Date: Mon, 7 Sep 92 21:37:05 CDT
From: klopfer@cerc.utexas.edu (Mike Klopfer)
Message-Id: <9209080237.AA23864@cerc.utexas.edu>
To: klopfer@natinst.com
Status: R


I am interested in reconstitution of membrane proteins into a bilipid layer.
More specifically bilipid membranes formed on something like a thin perforated
sheet of maybe some nonpolar material. If anyone could suggest some research 
work done along this line or even some protocols for reconstituting various 
photosynthetic units into such membranes it would be appreciated.

klopfer@natinst.com


Newsgroups: bionet.molbio.methds-reagnts
From: horton@molbio.cbs.umn.edu (Robert Horton)
Subject: Re: graft pcr, help needed********
Organization: University of Minnesota
X-Newsreader: Tin 1.1 PL5
Date: Wed, 16 Sep 1992 00:08:19 GMT

Rod Bonfiglioli (rbonfigl@waite.adelaide.edu.au) wrote:
: does anyone please have some information or protocols on "recombinant" or
: "graft" pcr....

Rod-

	Here are some references you might want to look at:

Horton, R.M., Hunt, H.D., Ho, S.N., Pullen, J.K. and Pease, L.R.
"Engineering hybrid genes without the use of restriction enzymes: 
gene splicing by overlap extension".  Gene 77:61-68, 1989.

Horton, R.M., Cai, Z., Ho, S.N., and Pease, L.R. 
"Gene splicing by overlap extension: tailor-made genes using the 
polymerase chain reaction". BioTechniques 8(5): 528-535, 1990.

Horton, R.M., and Pease, L.R. "Recombination and mutagenesis of DNA 
sequences using PCR". In: Directed Mutagenesis: A Practical Approach. 
(ed. M.J. McPherson) pp 217-247, IRL press, 1991.

Sarkar, G., and Sommer, S.S. BioTechniques (1990) 8:404.

I've got drawers full of other references, if you're interested.

Disclaimer:"of _course_ I'm tooting my own horn!" 8)
--
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: ser3@CUNIXF.CC.COLUMBIA.EDU (Stuart E Ravnik)
Newsgroups: bionet.molbio.methds-reagnts
Subject: SDS PAGE, additives and elutions
Date: 14 Sep 92 20:15:31 GMT
Distribution: bionet



Dear Net-folks,


I have two questions about different topics in running SDS gels.

1.  Has anyone ever heard of using 100mM N-ethylmaleimide in the SDS
buffer after doing immunoprecipitations to keep the IgG in the stack? 
The reference I have simply says "under these conditions, most
antibodies released from protein A by SDS migrated at >100kDa."  No
further protocols or methods were given.  Any ideas out there?


2.  Any good (read reliable, reproducible, and simple) mehtods for
eluting proteins in the basic range and about 30-40kDa from SDS gels?
I am running under reduced conditions in 12% acrylamide.


Any help you can give will be most appreciated.
You can e-mail me directly if you choose.


Thanks.

Stuart Ravnik    SER3@cunixf.columbia.cc.edu
From: jdboer@SOL.UVIC.CA (Johan deBoer)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Epstein Barr
Date: 4 Sep 92 23:27:03 GMT
Distribution: bionet







     Fellow moleculars,

     We are considering some work with  the Epstein Barr virus, and have  a
     few questions about it that we hope someone is able to answer.

     1)   What is the proper (latin  I guess) species, genus, organism etc.
          name for this virus?

     2)   The virus resides  in a cell line called MCUV5. What cell line is
          this, where does it come from, what does the name mean?

     3)   What are  the clinical  aspects, dangers,  risks, etc  associated
          with working with EBV?

     Any  comments, or pointers  to as where  the information  can be found
     would be greatly appreciated.

     Johan de Boer
     jdboer@sol.uvic.ca
     University of Victoria, BC
From: skspoidn@susssys1.reading.ac.uk
Newsgroups: bionet.molbio.methds-reagnts
Subject: ASAP
Date: 9 Sep 92 15:20:29 GMT
Distribution: bionet
Original-To: methods@uk.ac.daresbury

Sorry, I just deleted the message to which this 
is addressed so I have to post generally.

re Arctic Shrimp Alk Phos for dephos treatment:

Really? Arctic Shrimp?  I mean, how did they find out?
 Is there someone out there who tests different enzymes
 from different sources to see how good they are? 
(Have you tried the poodle S1 nuclease?) :)....

Oh, I have it! The BIG CHEESES worked this one out,
 and since BIG CHEESES only communicate with 
BIG CHEESES, I never got to hear of it.

Mike

Might not be science, but at least
 it's not about bionette.