HDTenEightyP/NetNews huggingface dataset:
From: browns@ccu.umanitoba.ca (Stuart Brown)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: (none)
Date: 28 Jan 93 19:07:47 GMT
Distribution: bionet
Organization: University of Manitoba, Winnipeg, Manitoba, Canada

In article <9301251402.AA17477@esds01.es.dupont.com> scolnipa@esvax.dnet.dupont.com 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?
>
>Pablo A. Scolnik

	I used to run really long gels in Dr. Yoder's lab a Cornell.  Instead
of using a gel dryer, we would open the two glass plates (with the gel 
sticking to the back plate-hopefully!), fix the gel on the plate in
Mehtanol/Acetic acid, and then dry the gel right onto the plate in a 95'C
oven.  You can then put your X-ray film right on the plate, cover with
another glass plate (so it doesn't move around) and expose for however
long you need in a light tight box. If your are really intent on getting
the top of the gel, just cut another piece of X-ray film and put it on top.
After you are done with the gel, strip it off the glass by soaking overnight
in 3M NaOH.

		-Stu

-- 
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
Newsgroups: bionet.molbio.methds-reagnts
From: saparici@crc.ac.uk (Dr. S.A.J.R. Aparicio)
Subject: Re: ABI sequencer: reaction clean-up ??
Reply-To: saparici@crc.ac.uk (Dr. S.A.J.R. Aparicio)
Organization: University of Cambridge, Department of Medicine
Date: Fri, 22 Jan 1993 15:48:58 GMT



In article <1993Jan22.122927.16338@news.unige.ch>, mike@medsun.unige.ch (michel morris) writes:
> 	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...)?


Is this part of the FAQ posting, if not perhaps it should be considered for
inclusion.

The vast majority of fluorescent ddNTPs are not incorporated in the PCR products
of cycle sequencing. If they are not removed effectively they form an enormous peak
at the start of a run and cause streak artifacts for several hundred bases afterwards,
thus seriously degrading the quality of the sequence. In our experience if you see even
traces of dye, by eye, then the sequence is usually so poor as to be unusable.

We do not use spin columns any longer - they are a nuisance to prepare and run and it is
often difficult to get consistent results - in our hands. The key here is consistency. There
are many methods which could remove the ddNTPs but the most reliable is one
developed by Andre Rosenthal and Steve Jones in our lab. This has now been fully published
and the reference is:

New Protocols for DNA sequencing with Dye Terminators,
Rosenthal A, Charnock-Jones DS
J. DNA Sequencing and Mapping; 1992; 3 : 61-64

Briefly, the method relies on using Sephadex G50 as a separation matrix but in static
columns of pre-determined size. The problem of flow is overcome by having triton X100
in the mixture (this seems not to affect the sequencing products). The details are int
the paper. I have had a lot of experience with this now and providing care is taken 
over the making up of the sephadex, the method is highly reproducible. The other caveat
is with lambda DNA, where I take 650ul of sephadex slurry rather than 600 - the large
amounts of high MW dna that come through sometimes bring through traces of ddNTPs as
well.


I hope this is of use to someone.

Dr Sam Aparicio
Molecular Genetics Unit
Dept. Medicine
Cambridge UK
From: RSCOTT@IRRI.CGNET.COM (RPSCOTT)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: RNA gels
Date: 26 Jan 93 10:45:34 GMT
Distribution: bionet

fm at "wsun@jeeves.ucsd.edu" writes:
>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?

I lifted the ff info from Curr Protocols in Molec. Biol. (but
these are based on DNA data--I guess ssDNA should migrate
comparably with RNA of equal length under denaturing condition):

===============================================================
  Acrylamide	   Bromphenol Blue	Xylene Cynanol
     (%)	   Marker  (b)		Marker (b)
---------------------------------------------------------------
      5                35                    130
      6                26                    106
      8                19                     75
     10                12                     55
===============================================================

I hope this helps.

------------------------------------
RScott
Intl Rice Res Inst
PO Box 933, 1099 Manila, Philippines
e-mail: rscott@irri.cgnet.com
------------------------------------
Newsgroups: bionet.molbio.methds-reagnts
From: rjhoffma@iastate.edu (Richard J. Hoffmann)
Subject: Re: Gilford Info
Organization: Iowa State University, Ames, IA
Date: Fri, 22 Jan 1993 03:23:17 GMT

I have an old Gilford 250.  I found the company to have been taken over
by CBI Corning (or something like that).  I'm home now and can't look up
the info, but if you haven'nt gotten what you need, email me and I'll
look it up tomorrow.

Dick
 
-- 
Richard J. Hoffmann                Internet: rjhoffma@iastate.edu
Department of Zoology & Genetics     Bitnet: rjhoffma%iastate.edu@isumvs
Iowa State University                 Voice: (515) 294-8075
Ames, Iowa 50011
From: bi__mvw@SELWAY.UMT.EDU (Michael van Waes)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Gilford Info
Date: 22 Jan 93 20:01:03 GMT
Reply-To: methods@net.bio.net
Distribution: bionet

>
>Sorry if this is a little out of the range of normal discussion on this
>newsgroup but I thought that some netter might have some information and
>be willing to help.
>
>We have an old GILFORD Stasar III spectrophotometer that needs some minor
>parts due to normal wear and tear.  The original papers are long gone and
>I have not been able to locate Gilford, Inc.   Anyone know what happened
>to them or more importantly, where (or if) the parts inventory may have
>been sold off to.
>
>Thanks for any comments.
>
>Mike
>
>
>************************************************************************
>*
>*  Michael Wooten
>*  Dept. of Zoology
>*  Auburn University
>*
>*  BITNET: mwooten@auducvax.bitnet
>*  INTERNET: mwooten@ducvax.auburn.edu
>************************************************************************
>
We currently own a Response II spec (also a Gilford), and I could give you
some advice before you buy a $16,000 machine...;-). Anyway, with regards to
your question, Gilford was bought by Ciba-Corning:

		63 North Street
		Medfield, Mass. 02052
		1-800-255-2121

           _________________________________________________________________
	   Michael van Waes	        |
	   Div. of Biol. Sci.	        |  "I never worry about the future, 
	   Univ. of Montana	        |       ... it comes soon enough. "
	   Phone: 406-243-5733  	| 
	   bi__mvw@selway.umt.edu	|            	Albert Einstein.
	   -----------------------------------------------------------------
From: sarahh@mbcr.bcm.tmc.edu (Sarah Highlander)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Gilford Info
Date: 22 Jan 93 21:18:13 GMT
Organization: Baylor College of Medicine, Houston, Tx
NNTP-Posting-Host: mbcr.bcm.tmc.edu

I had an old Stasar repaired by our in-house small electronics
shop (replaced the NIXI tubes that display the numbers). We 
obtained parts from Ciba Corning. Try 1-800-845-2666. I believe\
that I talked to Ken at extension x5352.

Good luck,

sarahh
From: GIETZ@bldghsc.lan1.umanitoba.ca
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re:pGEX fusion vector expression
Date: 25 Jan 93 18:49:00 GMT
Distribution: bionet

------------------------------------------------------------------------------
I have just cloned my gene into the pGEX vector using XL1-blue
cells.  Now the question is this:  is XL1-blue a suitable cell
line for expressing my fusion protein?  If not, what is a good
cell line to use?  Anyone who have had experience with the pGEX
vector please respond.

-fm

ps.  pGEX is a glutathione S-transferase fusion vector.
___________________________________________________

-fm
    We have used pGEX successfully for a number of genes
and have found that JM109 is the best strain for the production
of soluable fusions when compared to DH5alpha.  There was about
a 5-10 fold better production of full length fusion in JM109.  We have
also found that specific growth media also helps increase
the yield of active (properly folded?) protein.  
Blackwell and Horgan (1991) Febs letts 295;p10-12(Thanks to Kiran Madura)
describes a method using betaine in LB media with sorbitol to increase 
the yield of active( properly folded) fusion proteins.  We have tried it and 
it works very well for both our fusion proteins.

________________________________________
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: DMEIER@mis.mcw.edu
Newsgroups: bionet.molbio.methds-reagnts
Subject: SDS, nylon and hybridization
Date: 22 Jan 93 20:34:10 GMT
Distribution: bionet
Organization: Medical College of Wisconsin (Milwaukee, WI)

Hello to All!
  I am using Nytran nylon membranes in my Northern blotting. Often, I see 
SDS used in hybridizations, mostly with protocols using nylon filters. 
  Will anyone please share their expertise on this matter?  Is SDS
important in filter hybridizations, particularly with nylon?  Also, I've 
heard dextran works well with nitrocellulose, but that it is not 
recommended for nylon due to high background artifacts.
  Thanks much!

Dan
dmeier@mis.mcw.edu
From: bio1@navi.up.ac.za (Fourie Joubert)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Journals
Date: 21 Jan 1993 13:15:17 GMT
Organization: University of Pretoria
NNTP-Posting-Host: bio2.up.ac.za

Are there any ftp or e-mail sites available where journal databases may be 
accessed, something like ISI Current Contents? If you know of something like 
this, please mail me at bio1@navi.up.ac.za. 

Thanks in advance

Fourie Joubert
From: salbert@gwdgv1.dnet.gwdg.de ("GWDGV1::SALBERT")
Newsgroups: bionet.molbio.methds-reagnts
Subject: RE: pGEX fusion vector expression
Date: 24 Jan 93 17:21:11 GMT
Distribution: bionet
Original-To: methods@uk.ac.daresbury

-fm:
I have used pGEX cloning system and it worked just fine. In my procedure
E.coli DH5 alpha cells were used. But folks around me claims that XL-1
worked well for them.
The only possible drawback I assumed, that pGEX overproduces lac operator
(lac Iq gene product) itself. XL-1 strain overproduces the same protein
from its F'episome. So I was only affraid of over-over-production of lac Iq
gene product in XL-1 case (inhibition of cell growth, heterologous gene .
expression...??)
That's why I decided for DH5.

Hope it helps.
		Stefan
From: RSCOTT@IRRI.CGNET.COM (RPSCOTT)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: FISH Background
Date: 26 Jan 93 11:45:10 GMT
Distribution: bionet

Mike Morris at "mike@medsun.unige.ch" writes: 
>   We are doing fluorescent in situ hybridization on human chromosomes:
>single copy, painting, interphase, the lot.
>   Although we have no trouble (at last!) getting good signals and good
>specificity, we very often have a significant "halo" of cytoplasm around
>metaphases, which fluoresces slightly (with biotin-labelling, avidin
>development). I suspect that this may be due to cellular biotin picking
>up the label.

If guess if it's really cellular biotin that's been causing mischief in 
your FISH I suggest you try digoxigenin labelling. Unlike biotin which
is rather ubiquitous, digoxigenin (a steroid appended as a moiety to dUTP)
has only been found to date in the plant Digitalis purpurea. The
detection  I know for DIG is an ELISA based on alkaline phosphatase
which can also use a several fluorescent substrate systems. Boehringer
Mannheim (Germany) has been developing the DIG system. We have been using
their kit for our RFLPs for quite some time now and they work neatly. 
(I just hope I don't create an impression that I'm an agent of BM.)


R.P.Scott
Genet Lab
Plant Pathol Div
Intl Rice Res Inst
PO Box 933, 1099 Manila, Philippines
e-mail:  rscott@irri.cgnet.com
From: ming@ZEUS.AHABS.WISC.EDU ("Ding Ming")
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: photography
Date: 28 Jan 93 16:35:28 GMT
Reply-To: ming@zeus.ahabs.wisc.edu
Distribution: bionet
Organization: Animal Health & Biomedical Sciences


>Sender: news@newssrv.edvz.univie.ac.at (News System - Vienna 
>University)
>Nntp-Posting-Host: helios.edvz.univie.ac.at
>
>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
>
>
Kodak Tri-X Pan and Plus-X Pan will do the job, the only additional 
things you need are a macro lense and a red filter.

ding



~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ 
    Ding Ming                     
    (608)265-3544                   ming@calshp.cals.wisc.edu
    Sweet Protein Section           ming@zeus.ahabs.wisc.edu
    Taste Research Lab               
    Department of Animal Health & Biomedical Sciences    
    University of Wisconsin-Madison, 1655 Linden Dr., Madison, WI53706      
~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ 
    Office Location: A215 Babcock Hall   HomePhone: 233-2159      
~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~
From: cytogen@chmeds.ac.nz (Martin Kennedy)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: insert-positive blue cols
Date: 24 Jan 93 22:27:22 GMT

In article <1993Jan21.090729.79@ebc.ee>, mspeek@ebc.ee (Mart Speek) writes:
> 
> 
> I would like to know whether anyone has encountered problems
> in traditional blue/white colony or plaque screening procedures
> (beta galactosidase assay) when sorting out (non)recombinant
> clones. My question is :-( Could relatively long inserts (>0.5kb)
> sometimes give rise blue (beta gal+) clones? How is it possible?
> Any theoretical or practical solutions (incl refs) to this 
> particular problem are welcome.
> 
> M. Speek
> 
> mspeek@ebc.ee (ESTONIA)

The blue/white assay is based on disruption of disruption of part of the coding
sequence for lacZ by insertion of the cloned DNA fragment.  Any fragment which
is a multiple of 3 bases long and does not contain one of the stop codons (TAA,
TGA and TAG - I think) can allow formation of a "tripeptide" fusion protein
that may still be enzymatically active.  I've had blue plaques from an insert
that was 300bp long for this reason.  Furthermore, any cloned insert that
contains a start codon inframe with the downstream lacZ open reading frame may
also give rise to "blue" plaques/colonies.  In effect, there can be a continuum
in the colour of recombinant clones, from white through to blue.  If you plate
your putative recombinant on X-gal next to a true wild type lacZ vector, you
may be able to detect a difference in the intensity of the blue colour.

All this sort of stuff is thoroughly described in a number of articles by Jo
Messing, but two of the best are:

Messing, J. (oops - don't know the year) New M13 vectors for cloning . Methods
in Enzymology 101, 20-79   (Everything you ever wanted to know about M13 but
were afraid to ask Bionet)

Messing, J. (1991).  Cloning in M13 phage or how to use biology at its best. 
Gene 100, 3-12    (A delightful and informative historical review of the
development of the M13 and pUC vectors - recommended reading for any student of
molecular biology).


Cheers,

Martin

Martin A Kennedy (E-mail = cytogen@chmeds.ac.nz)
Cytogenetic and Molecular Oncology Unit
Christchurch School of Medicine
Christchurch, New Zealand
From: GIETZ@bldghsc.lan1.umanitoba.ca
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: fusion protein expression vectors
Date: 21 Jan 93 22:14:00 GMT
Distribution: bionet


%%%%%%%%%%%%%%%%(Brian J.Baumgartner) writes:%%%%%%%%%%%

>We would like to create fusion proteins for synthesis of antibodies.  We
>must be able to cleave our desired peptide from the fusion product and
>affinity purify the fusion.  Several companies make these types of
>prokaryotic expression vectors.  We would appreciate any comments on which
>vector has proven the most effective for you.  No sales reps please!
>
>Thanks, 
>
>Brian Baumgartner
>Dept. of Biochemistry
>Baylor College of Medicine
>Houston, Tx. 77030
>e-mail: brianb@mbcr.bcm.tmc.edu
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%

Hi Brian!
    We have used pGEX-2T system, which fuses your gene
to the glutathione S transferase protein and has a thrombin cut
site between this protein and yours, with great success.  See
Smith and Johnson (1988) Gene 67,31-40.  The proteins can be
affinity purified using glutathione agarose in a single step to 
a very high level of purity.  Our fusion protein can be cut directly 
'from the beads with a very high efficiency.  The paper by Oettinger
et al (1992) Biotechniques 12;p544, describes using this system for
the production of antisera.  In addition we have found that the growth
conditions are important for the production of maximum amount of
fusion proteins.  Our best are 25oC growth with2 hr induction with IPTG in 
strain 
JM109.  We have also found that specific growth media also helps increase
the yield of active (properly folded?) protein.  Blackwell and Horgan (1991) 
Febs letts 295;
p10-12.(Thanks to Kiran Madura) describe a method using betaine in LB media
 with sorbitol to increase 
the yield of active( properly folded) fusion proteins.  We have tried it and 
it
works very well for  both our fusion proteins.  pGEX is marketed by 
Pharmacia/LKB.

Hope this helps!

Dan Gietz
U of Manitoba
GIETZ@BLDGHSC.LAN1.UMANITOBA.CA
From: rander@cco.caltech.edu (Roger F. Anderson)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Lambda Sequencing; If I can You can!
Summary: Lambda sequencing is easy if you know the tricks.
Keywords: sequencing
Date: 26 Jan 93 22:42:01 GMT
Organization: California Institute of Technology, Pasadena
NNTP-Posting-Host: sandman.caltech.edu

everal years ago (I hate to admit it but I am in my 6th year of graduate school)
I needed to clone the members of an actin family, some of you out there are
now realizing who I am. Anyhow rather than subclone and sequence all of these
clones I had I had primers made to conserved regions and figured out how
to sequence right off of the clones. It works for 100 -250 bases depends on
alot of factors. 
1) initiating extensions on ICE.
2) shart annealing times
3) clean DNA
4) double concentration of nukes (I always use double USB recommended)
5) Pharmacia nukes USB nukes are crap!
6) Sequence some sequence goes back together very fast.

If anyone needs a protocol let me know. I say needs because it is too long
to post and I can't spend all day at the fax machine. I guess you'll have
to tell me why you need it.
Thanks for your time
ROG
Newsgroups: bionet.molbio.methds-reagnts
From: news@news.yale.edu (USENET News System)
Subject: re: Cloning PCR fragments
Organization: Yale University
Date: Fri, 22 Jan 1993 17:36:16 GMT


In article <9301201721.AA25989@socrates.ucsf.EDU> elder@CGL.UCSF.EDU (Bruce Elder) writes:
>
>In response to my response to Yin Chai's questions about PCR cloning I have
>received many inquiries about why pDK101 is so special a vector. I don't really 

>(about 2 ul when I do it). The cut vector keeps for about 3-4 months, but this
>is somewhat variable.
>
>Bruce Elder
>elder@cgl.ucsf.edu

Can you tell us how we cn get this vector?

Gary Rudnick
Rudnick@biomed.med.yale.edu
From: MCRGILAD@VM.UOGUELPH.CA (Gilad Bernadsky)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Short peptide sequencing
Date: 28 Jan 93 22:50:30 GMT
Distribution: bionet

Hi,
I am sending this message for a respectful graduate student in our lab.
Please reply to my E-mail address (mcrgilad@vm.uoguelph.ca).

Thx,

Gilad
Dept. of Microbiolgy
University of Guelph
Canada
------------------------Original message-------------------------
HELLO,
I AM HAVING DIFFICULTIES SEQUENCING A SHORT (24 AMINO ACIDS)
HYDROPHILIC PEPTIDE BY CONVENTIONAL MEANS (I.E. SOLID SUPPORT
AND GAS PHASE).  I SUSPECT THE PEPTIDE DOES NOT BIND TO THE
SAMPLE MEMBRANE (WE TRIED THE MILLIPORE SEQUENET KIT).
ANY SUGGESTIONS AND IDEAS WOULD BE MUCH APPRECIATED.
THANKS,
MARK BRAY

DEPT. OF MICROBIOLOGY
UNIVERSITY OF GUELPH
CANADA
Newsgroups: bionet.molbio.methds-reagnts
From: browns@ccu.umanitoba.ca (Stuart Brown)
Subject: Re: Colony Hybridisation with biotinylated oligo
Organization: University of Manitoba, Winnipeg, Manitoba, Canada
Distribution: bionet
Date: Fri, 22 Jan 1993 21:14:00 GMT

In article <1993Jan20.145525.17865@gserv1.dl.ac.uk> ahill@crc.ac.uk (Mr. A.F. Hill) writes:
>
>
>I'm trying to set up a system for doing colony hybridisations using a
>biotinylated oligo (23-mer) and using streptavidin-biotin-alkaline phosphatase
>and NBT/BCIP to get the colour reaction...
>I've spotted the oligo onto a filter and can read a signal down to 10pg but
>when i hybe with the oligo and develop the colour i get a nice purple
>membrane...
>Andy

	Sounds to me like you need better blocking.  Some kits come with
a "Secret Blocking Reagent" which is probably BSA.  Also, you can try
pre-hybridizing with a mixture of salmon sperm and plasmid without insert.

Colony blotting is a very dirty proceedure. I've also read of a technique
where a library is subdivided by plating transformed bacteria on a large
number of plates (maybe 1000 to 10,000 clones per plate), then the colonies 
from each plate are pooled for a large scale plasmid prep (keeping a replica,
of course).  The plasmid pools are then tested by dot blot hybridization.
Pools with positive signal are sudivided and the proceedure repeated, etc.

	-Cheers  -Stu
-- 
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: RSCOTT@IRRI.CGNET.COM
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: breaking small vols S. pombe cells
Date: 23 Jan 93 06:40:16 GMT
Distribution: bionet

David Salt at "BOTSALT@vm.uoguelph.ca" writes:
>I need to break small volumns of S. pombe (fision yeast), approx 1 ml, in
>the presence of 100mM HCl to extract the acid soluble thiols.
>I have tried grinding with no luck. Any other ideas.

Why don't you try lyticase to digest the cell wall first before
you perform acid extraction. Alternatively, you can perform
cell disruption with glass beads as recommended earlier by
Dan Geits of the U. of Manitoba.

***See Current Protocols in Molec. Biol.
***under Chapt. 13: Saccharomyces cerevisiae

===========================================================
Z. Scott
Intl Rice Res Inst
e-mail: rscott@irri.cgnet.com
===========================================================