HDTenEightyP/NetNews huggingface dataset:
From: msw1633@zeus.tamu.edu (Mark S. Whitsitt, N5RJF)
Subject: Extinction Coefficient
News-Software: VAX/VMS VNEWS 1.41    
Reply-To: MSW1633@SUMMA.TAMU.EDU
Organization: Texas A&M University, Biochemistry Department
Date: Fri, 20 Nov 1992 15:41:00 GMT

Does anyone know the molar extinction coefficient of 
o-nitrophenol-B-galactoside (ONPG)?  I need this for one of my students who is 
trying to empirically determine the equilibrium constant for beta-galactosidase.

Thanks 

Mark S. Whitsitt, N5RJF        Texas A&M University, Dept of Biochemistry
Internet:  msw1633@summa.tamu.edu 	  College Station, Tx. 77843-2128
AMPRnet:   n5rjf@n5rjf.ampr.org 			   (409) 845-0832
Packet/APLINK:  N5RJF @ KE5HE.TX.USA.NA
   "You can't throw darts when you're empty, man." - another Shadelism
From: msw1633@zeus.tamu.edu (Mark S. Whitsitt, N5RJF)
Subject: Re: RNA from seeds
News-Software: VAX/VMS VNEWS 1.41    
Reply-To: MSW1633@SUMMA.TAMU.EDU
Organization: Texas A&M University, Biochemistry Department
Distribution: bionet
Date: Thu, 19 Nov 1992 18:53:00 GMT

In article <921118180531_76467.702_DHE72-4@CompuServe.COM>, 76467.702@CompuServe.COM (Alan Gerstein) writes...
>he is trying to isolate mRNA from tobacco seeds. he has already tried two
>classes of methods: guanidinium and phenol. basically what happens is that
>everything turns an ugly kind of brown and is very sticky.


Also see Shirras and Northcote (1984) Planta 162:353-360

I have had similar problems with RNA and brownish contamination preventing 
efficient mRNA isolation.  The RNA extraction protocol here has solved at least 
my mRNA problem.  The most helpful step is the washing of the RNA pellet with 
3M NaOAc pH 5.5. this has removed any of the nasties from the prep.

Good Luck,

Mark S. Whitsitt, N5RJF        Texas A&M University, Dept of Biochemistry
Internet:  msw1633@summa.tamu.edu 	  College Station, Tx. 77843-2128
AMPRnet:   n5rjf@n5rjf.ampr.org 			   (409) 845-0832
Packet/APLINK:  N5RJF @ KE5HE.TX.USA.NA
   "You can't throw darts when you're empty, man." - another Shadelism
From: u6064043@ucsvc.ucs.unimelb.edu.au
Newsgroups: bionet.molbio.methds-reagnts
Subject: Help needed: genomic DNA isolation
Date: 17 Nov 92 07:03:59 GMT
Organization: The University of Melbourne

Hi netters,

I've been trying to isolate plant genomic DNA from leaf for making a
genomic library. The procedure I used is quite straight forward: grind
leaf, extract, collect nuclei by centrifugation, then lyse by 2% CTAB,
phenol/chloroform extract twice, ethanol precipitate DNA, redissolve
in 20 mM Tris pH8, 10 mM EDTA overnight at 4C, followed by another
phenol/chloroform/ethanol ppt. and redissolve in TE. I've been able
to get genomic DNA >25Kb (can't tell exact size by agarose gel) without
visible RNA contamination (I include RNase A in the extraction buffer).
However, the biggest problem with this method is the difficulty in
redissolving DNA after ethanol ppt. Typically, the pellete won't dissolve
overnight or by gentle stirring of the tube. I had to centrifuge to get
rid of the insoluble stuff and thus get very low yield (15 gram leaf
---> 50 ug DNA). I've also tried to enhance the solubility by heating at
50C for a few hours without obvious effect. Any suggestions?
Thanks in advance

Du
From: wmelchior@ntet.nctr.fda.gov
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: dye removal from ABI terminator reactions.
Date: 23 Nov 92 10:56:37 -0600
Distribution: bionet
Organization: NATIONAL CENTER FOR TOXICOLOGICAL RESEARCH

> We now routinely purify the excess dye from reactions using home (lab)-made
> quick spin columns.  They may not overcome the tedium but they sure are 
> cheap.  We make a hole in the bottom of 0.5 ml eppendorf using a 26 g needle
> and add about 25 ul volume of zirconium glass beads.  Then I pour sephadex
                                ^^^^^^^^^
> G-50 in 0.3M NaAc (5g in 60 ml) to the top of the tube.  Stick the small
> eppendorf inside a 2 ml tube and spin at about 500 rpm for couple of minutes.
> Now the tube is transferred to a new 1.5 ml tube and the sample is added on
> top.  Now I spin at about 1500 rpm for couple of minutes.  
> The eluate is precipitated by atleast 3 volumes of isopropanol washed with
> 70% etoh and resuspended for loading.

Raj-
Thanks for the method.  One question: why _zirconium_ glass beads?  
Wouldn't regular glass work as well?  And what size beads do you use?
________________________________________________________________________________
The opinions stated are mine, not those of NCTR or its sponsoring organizations.

Bill Melchior                                ||     OMNISCIENCE
National Center for Toxicological Research   ||    Knowing what
Jefferson, AR  72079                         ||    thou knowest not
(501) 543-7206                               ||    is in a sense
                                             ||    omniscience.
WMELCHIOR@NTET.NCTR.FDA.GOV                  ||       from Grooks, Piet Hein
From: srckbjg@lincoln.cri.nz
Newsgroups: bionet.molbio.methds-reagnts
Subject: Removal of Bind-silane
Date: 18 Nov 92 22:58:14 GMT
Organization: Canty Ag & Science Centre, New Zealand.

Hi,

Previous inhabitants of our lab favoured the use of 1850-251 Bind-Silane
(gamma-Methacryloxypropyltrimethoxysilane) made by LKB Bromma to bind
polyacrylamide sequencing gels to the large glass plate, so that you couln't
lose it during fixing etc.  We no longer bother with this method, but
unfortunately most of the plates in the lab are bind-silane treated, and can't
be used for normal sequencing.  So whats the best (and safest) way of
removing the bind-silane from these plates?

Our few untreated plates are getting very battle-worn so your suggestions
would be greatly appreciated.

Thank you

Brent Gilpin
Crop & Food Research
Lincoln
New Zealand
From: djt2@po.CWRU.Edu (Dennis J. Templeton)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Removal of Bind-silane
Date: 19 Nov 1992 18:20:06 GMT
Organization: Case Western Reserve University, Cleveland, OH (USA)
Reply-To: djt2@po.CWRU.Edu (Dennis J. Templeton)
NNTP-Posting-Host: thor.ins.cwru.edu


In a previous article, srckbjg@lincoln.cri.nz () says:

>Hi,
>
>Previous inhabitants of our lab favoured the use of 1850-251 Bind-Silane
>(gamma-Methacryloxypropyltrimethoxysilane) made by LKB Bromma to bind
>polyacrylamide sequencing gels to the large glass plate, so that you couln't
>lose it during fixing etc.  We no longer bother with this method, but
>unfortunately most of the plates in the lab are bind-silane treated, and can't
>be used for normal sequencing.  So whats the best (and safest) way of
>removing the bind-silane from these plates?
>
>Our few untreated plates are getting very battle-worn so your suggestions
>would be greatly appreciated.
>
>Thank you
>
>Brent Gilpin
>Crop & Food Research
>Lincoln
>New Zealand
>

We still like this method, but buy the cheap material from Sigma (it's a
paint additive, I'm told, and is really marked up by LKB)

To re-use our plates, we hydrate the gel and plate in wqater for a few
minutes, then scrape the gel onto a paper towel with a putty knife.  We
then stack the plates in a developing tray (using gel spacers to keep them
separated, and let them soak for an hour or overnight in 0.5 N NaOH, then
rinse and put away as clean.  The base will effectively remove the bind
agent.

good luck

dennis
Newsgroups: bionet.molbio.methds-reagnts
From: pnh@fcsparc6.ncifcrf.gov (Paul N Hengen)
Subject: Re: Looking for small vector
Organization: Frederick Cancer Research and Development Center
Distribution: bionet
Date: Sat, 21 Nov 1992 00:36:01 GMT

LEES@SASK.USASK.CA writes:

>If does anyone have information about small vector which has 
>both ampicillin and kanamycin markers with less than 3Kb,
>please let me know.

On the plasmid pNH-Kan/oriT, there is a 1.4 kb kanamycin resistance
gene that can be easily subcloned onto an ampicillin resistant plasmid.
If you were to do this on any pUC vector, you would have a 1.4 + 2.7 kb
construct. That's more than you care for, but the omega fragment may
be useful to you. Is there a reason for it being less than 3 kb?

The reference is:

@ARTICLE{Hengen1992.oriT,
AUTHOR = "P. N. Hengen
        and V. N. Iyer",
TITLE = "DNA cassettes containing the origin of transfer (oriT) of two
        broad-host-range transfer systems",
JOURNAL = "Biotechniques",
YEAR = "1992",
VOLUME = "13",
PAGES = "57-62"}

Abstract:

Plasmid constructs are described which carry retrievable DNA cassettes
containing the origin of transfer region (oriT) from two broad-host-range
plasmids. Restriction of these high copy number plasmids with any one of a
variety of enzymes yields a linear DNA fragment of convenient size
containing the oriT region of either pCU1 or RK2. This DNA can be
ligated into any vector or recombinant plasmid containing a compatible
enzyme site and can be easily identified by size on an agarose gel.
Any plasmid can therefore be mobilized using a number of helper strains or
conjugative plasmids derived from the parental plasmids. In addition, the
cassettes can be used for a variety of genetic manipulations including
"selectable" linker mutagenesis.


Paul N. Hengen
National Cancer Institute
Frederick Cancer Research and Development Center
Frederick, Maryland 21702-1201 USA
From: bsh@MED.PITT.EDU (Basavaraju Shankarappa)
Newsgroups: bionet.molbio.methds-reagnts
Subject: dye removal from ABI terminator reactions.
Date: 19 Nov 92 22:26:11 GMT
Distribution: bionet

From: scarr@kean.ucs.mun.ca
Subject: Removing dye from 373A reactions

>Has anyone found a cheap, quick, reliable way of removing excess dye
>terminators from a Cycle Sequencing reaction for an ABI373A sequencer?
>We usually use an isopropanol extraction, but this seems to leave
>variable amounts of the dyes behind. The ABI recommended BioSpin columns
>do a better job, but they are costly and the ritual is slow & tedious.
>Anyone had any experience with other commerical columns?
>Steve Carr 

We now routinely purify the excess dye from reactions using home (lab)-made
quick spin columns.  They may not overcome the tedium but they sure are 
cheap.  We make a hole in the bottom of 0.5 ml eppendorf using a 26 g needle
and add about 25 ul volume of zirconium glass beads.  Then I pour sephadex
G-50 in 0.3M NaAc (5g in 60 ml) to the top of the tube.  Stick the small
eppendorf inside a 2 ml tube and spin at about 500 rpm for couple of minutes.
Now the tube is transferred to a new 1.5 ml tube and the sample is added on
top.  Now I spin at about 1500 rpm for couple of minutes.  
The eluate is precipitated by atleast 3 volumes of isopropanol washed with
70% etoh and resuspended for loading.

	I intend to have large number of colmumns poured and stored at 4 C.
I think it can be done.  But I havenot done it yet.  This way, all I have 
to do is do a quick spin, exchange the tubes add sample spin and ppt.
I get my zirconium beads from Biospec products PO Box 772 Bartelsville
OK 74005.  (918)-336-3363.  Cat # 11079-101. $20.00 a pound; lasts for a
life-time. 
Raj Shankarappa
bsh@med.pitt.edu
Univ of Pittsburgh

Newsgroups: bionet.molbio.methds-reagnts
From: jgraham@bronze.ucs.indiana.edu (the End)
Subject: Re: kits and kits and kits and mutagenesis
Organization: Indiana University
Distribution: bionet
Date: Mon, 16 Nov 1992 00:35:47 GMT


Generating wild type clones via the KUNKEL procedure (whichever kit that is :)
in the absence of mutagenic primer is usually a problem of self priming in the
single-stranded DNA prep. Be sure you are generating good single stranded
phagemid yields by titering the superinfection supernate for amp resistant
transducing particles (1 x10-12 is good ).

Three things you should try in decending order:

1. Kunkel's Methods in Enzmology paper (154 p.367) indicates that after 
precipitating the phage or phagemids with PEG, resuspend them in TE buffer
with vigorous vortexing/pipetting. Then after standing on ice 10 min, spin
them at full speed in your microfuge and discard the pellet. If you do this 
in glass, you will notice the supernatant is a blue-ish clear solution rather
than a cloudy grey. Extract with phenol (no chlorform) and then phenol-CIA or
just CIA. Continue with an AMMONIUM ACETATE ethanol precipitation. Oh yeah,
do the phage precip with NH4Oac-PEG.

2. If this doesn't work, be sure to spin the supernatant containing the 
phagemids in the intiial superinfection culture 2 times for 10 min in the
microfuge at 14,000. This is necessary to remove all plasmid (wt) containing
cells from the super.

3. Gel purify the single stranded DNA template. Do not get RNAse or DNAse
anywhere near you preps at any point, even though this is suggested in one of
the popular KITS. You will generate millions of tiny primers that will be 
carried along with your single stranded DNA.


If all this fails, then do your annealing reactions in screw-capped eppies
that have been submerged under water in a beaker with 800 ml of hot (85) water.
Place this beaker in a 90 oven for an hour, and then gradually reduce the oven
temperature setting.

This should get you there, but there are of course other perils. Watch the 
primer to template ratio if you have any degenerate positions in the mutagenic
primer. Also watch out for transformants who have not fully segregated the
possible mutant plasmids.


Good luck,

Jim
J. Graham
Biology and Chemistry Depts.
Indiana University -Bloomington
From: obrien@molbio.cbs.umn.edu (Darin O'Brien)
Newsgroups: bionet.molbio.methds-reagnts
Subject: deblocking
Date: 18 Nov 92 21:19:37 GMT
Organization: University of Minnesota
X-Newsreader: Tin 1.1 PL5

I have an oligo given to me by someone else.  Unfortunately, the oligo was not
deblocked and nobody around here knows how to do it.  Does anyone out there know
how to do this?  I don't know what solution the oligo is in now.  Can I
just precipitate the oligo and treat overnight in ammonium hydroxide?  Any help
would be greatly appreciated.

darin



         _______________________________________________________
      |                                                        |
      |  Darin O'Brien        Email: obrien@molbio.cbs.umn.edu |
      |  5-110 Moos Tower     Phone: (612) 624-9663            |
      |  University of        Fax: (612) 626-7031              |
      |  Minnesota                                             |
      |  515 Delaware St. S.E.                                 |
      |  Minneapolis, MN 55455                                 |
      |________________________________________________________|
From: u6064043@ucsvc.ucs.unimelb.edu.au
Newsgroups: bionet.molbio.methds-reagnts
Subject: bo
Date: 19 Nov 92 15:10:23 +1100
Organization: The University of Melbourne

Hi,

Thanks for all the advices and protocols I've receviced for the last two days.
I'm surprised by the number of the responses my original post generated, and
am convinced that this is a great newsgroup where one can get real help.
Anyway, I'm sorry that I couldn't reply to everyone, here is a brief summary
of the advices I've received.
(1) ppt with isoproponal and fish out DNA with glass rod etc.
    I've tried this method, however didn't help in my case.
(2) don't completely dry DNA after ppt.
    good suggestion. However, this can't be my problem as I only dry pellete
    by standing on the bench for a few minutes then suspend in buffer.
(3) polysaccharides content causing the insoluble problem.
    very likely. As I used green leaves (not etiolated) which have lot of
    starch. Starch will co-ppt with nuclei and thus causing problems.
So next step, really, is to avoid the contamination of polysaccharides during
DNA isolation.

Du

Newsgroups: bionet.molbio.methds-reagnts
From: rmkdhhp@ucl.ac.uk (Dr Huaizheng Peng)
Subject: PCR for TCR
Original-To: methods@uk.ac.daresbury
Distribution: bionet
Date: Thu, 19 Nov 1992 09:14:22 GMT

Dear Netter,

Does anyone have any experience of using PCR to show clonal rearrangement of T cell receptor genes in T cell neoplasia? If so, which methods are most reliable?

Thank you in advance.

Dr. Peng HZ
Histopathology
UCL Medical School
London
Newsgroups: bionet.molbio.methds-reagnts
From: mimq@hippo.ru.ac.za (Molapo Qhobela)
Subject: Cloning vector
Keywords: Mob
Organization: Rhodes University, Grahamstown, South Africa
Date: Mon, 23 Nov 1992 09:56:29 GMT


HELP!  HELP!  HELP!  HELP!  HELP!
I am looking for a plasmid with the following:
1) Mob+
2) Two resistance markers - one with unique restriction/cloning   
   site/s.
3) Cole1/pMB1/pACYC replicon.
4) 8 kb or smaller.


-- 
   Molapo Qhobela PhD.                |  Do not ever get caught with 
   Rhodes University - Microbiology   |       your plants down!!
   Internet: mimq@hippo.ru.ac.za      |  Consult a Plant Pathologist 
   Telefax: +27 (0461) 24377          |            via APS
From: pouliot@GENETHON.GENETHON.FR (Yannick Pouliot)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Address of European Cell Line Repository?
Date: 23 Nov 92 09:21:10 GMT

Fellow netters, can some good soul list the address/phone number of the European equivalent of the American Type Culture Collection (ATCC)?

Danke Schon,

Yannick Pouliot
Genethon
Newsgroups: bionet.molbio.methds-reagnts
From: cenderli@nyx.cs.du.edu (C. S. Enderlin)
Subject: Re: Removal of Bind-silane
Organization: Nyx, Public Access Unix @ U. of Denver Math/CS dept.
Distribution: bionet
Date: Mon, 23 Nov 92 21:58:00 GMT

After stripping the plate with NaOH, how about applying
no-bind silane or something like SigmaCote?
From: jhu@RESUNIX.RI.SICKKIDS.ON.CA
Newsgroups: bionet.molbio.methds-reagnts
Subject: Request for the plasmid pKK240-11
Date: 24 Nov 92 05:07:45 GMT
Distribution: bionet

Hi Everybody:
        I would appreciate it very much if someone could send me the
plasmid pKK240-11 or its derivative.  I would like to use the tac promoter
to express His-tagged proteins in E. coli.  I have some proteins expressed
from the T7 promoter, but the protein levels are much lower than those
expressed from GST-vectors.    
        Thank you very much in advance.
Jim Hu
Research Institute Rm 8134
Hospital for Sick Children
555 University Avenue
Toronto, Ontario, M5G 1X8
Canada
Phone:416-813-6429
FAX:  416-813-5085
From: SHANELT@BIOLAN.UNI-KOELN.DE (Sabine Hanelt)
Newsgroups: bionet.molbio.methds-reagnts
Subject: nonradioactive northerns
Date: 17 Nov 92 14:15:17 GMT
Distribution: bionet
X-Vmsmail-To: smtp%"methods@daresbury.ac.uk"
Original-To: methods@uk.ac.daresbury



Hi netters,
my problem is how to get a nonradioactive northern blot.
I tried already Digoxigenin DNA and RNA probes (Boeringer System)
and also biotinylated DNA probes (USB Gene Images). None of these
systems seem to be helpfull, maybe I make a general mistake.
Has anyone experiences with nonradioactive northern blots that are not
so frustrating like mine?

Thanks for your help
Sabine
Newsgroups: bionet.molbio.methds-reagnts
From: scarr@kean.ucs.mun.ca
Subject: Removing dye from 373A reactions
Organization: Memorial University. St.John's Nfld, Canada
Date: Thu, 19 Nov 1992 19:01:32 GMT

Has anyone found a cheap, quick, reliable way of removing excess dye
terminators from a Cycle Sequencing reaction for an ABI373A sequencer?
We usually use an isopropanol extraction, but this seems to leave
variable amounts of the dyes behind. The ABI recommended BioSpin columns
do a better job, but they are costly and the ritual is slow & tedious.
Anyone had any experience with other commerical columns?

Thanks for the input

Steve Carr
From: skspoidn@reading.ac.uk
Newsgroups: bionet.molbio.methds-reagnts
Subject: UDNA mutagen
Date: 16 Nov 92 09:42:02 GMT
Distribution: bionet
Original-To: methods@uk.ac.daresbury

re 
Dear netters,

I am trying to do site-directed mutagenesis using the Bio-rad
kit.  I am using CJ236 cells and my clone in the Bluescript
plasmid to make SS DNA with VCM13 helper phage.  Following the
protocol, I do a negative control where I add everything except
the mutagenic primer.  However, when I transform XL1-blue cells
with this negative control DNA, I get colonies on the plate.  In
addition, when I screened the colonies from my positive plate, I
don't get any mutants, only wild type.  Can anyone out there
have had this problem and could offer some
explanation/solutions?
------

Whatwedowhenwehavethisproblem:

1. Buy a new kit!!! (hee hee, only joking)
2. Do you titrate your UDNA on CJ's and your normal bugs? 
you should get a 10000 fold difference in titre/plasmid. You may 
find that your initial input of SS DNA is too high, and is
 contaminating the UDNA.
3. Use as little DNA pol as possible. We use G32 protein and
 DNA pol, and find that even slight excess of the pol causes 
wierd things to happen, including primer dislocation.
4. Is your helper phage helping? ie are you getting Bluescript
 out in the end. Cant help with optimizing that bit of the
 protocol, as we use M13. Note that this makes UDNA titration easy too.

Mike Poidinger 
Dept of Microbiology
University of Reading
From: Klaus.Matthaei@anu.edu.au
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Result from "Primer" programme
Date: 22 Nov 92 22:27:36 GMT
Distribution: bionet

>I have been using the "Primer" programme on the published sequence of a mouse
>gene (HSP70.1) and it reports homology in the 3' non-transcribed region, with
>the mouse repetitve element B2.
>My question is, could this explain why my attempts at using a probe which
>is derived from the 3' region of this gene, has failed so far in Southern
>blots - all I get is hybridization to apparently all DNA, even when washed
>at 65 degrees in 0.1xSSC.
>Does anyone know HOW homolgous the sequence has to be before Primer reports
>it ?
>
>Any comments appreciated,
>Sean

Hi Sean

My guess is that the homology with the B2 element is your problem.  0.1xSSC
at 65 is certainly stringent enough to ensure that only true hybrids
remain.  However, in my experience even a 20bp region of total homology
with a repeat element is enough to turn your entire lane black.  Is it
possible to remove the region of homology?

I 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

"If all else fails : Read the instructions"
--------------------------------------------------------------------------
From: afc@gnv.ifas.ufl.edu
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Need methods for molecular diversity
Date: 20 Nov 92 16:58:36 -0500

In article <98347@netnews.upenn.edu>, btozier@pobox.upenn.edu (William A. Tozier) writes:
-deleted-
>   As a specific question: Is anyone aware of an electrophoretic technique that
> would discern DNA, RNA and protein simultaneously?

I used a stain called "Stainsall" (or something like that) in the mid-70s.
It stained DNA, RNA, and protein, each a different color.

Andrew Cockburn
USDA
 
> Thanks for your help,
> Bill Tozier
> Biology Department
> University of Pennsylvania
> 
> btozier@pobox.upenn.edu
> bill@amy.med.upenn.edu
Newsgroups: bionet.molbio.methds-reagnts
From: mulligan@kean.ucs.mun.ca (Martin E. Mulligan)
Subject: Multi-Pol DNA Sequencing System
Organization: Memorial University of Newfoundland
Date: Thu, 19 Nov 1992 13:09:22 GMT

Has anyone ever used the Multi-Pol DNA Sequencing System sold by
Clontech?  Can you recommend it or otherwise?  In particular, how
does it compare with Sequenase?  By the time we pay for extra dry ice
and shipping, Sequenase kits become prohibitively expensive here - I'd
like to find a reliable alternative and would be interested in other
labs' experiences with other sequencing systems.

Thanks,

Martin E. Mulligan
Dept. of Biochemistry, Memorial University of Newfoundland

email: mulligan@kean.ucs.mun.ca