HDTenEightyP/NetNews huggingface dataset:
From: ray@uk.ac.le ("Dr R. Dalgleish")
Newsgroups: bionet.molbio.methds-reagnts
Subject: Tracking dye for alkaline agarose gels?
Date: 21 Aug 92 12:30:29 GMT
Distribution: bionet
Original-To: methods@uk.ac.daresbury
X-Mailer: ELM [version 2.3 PL0 (LUT)]

Do anybody know of a tracking dye that can be used with alkaline agarose gels
that will not diffuse quickly out of the gel? Both bromocresol green and
eriochrome black T diffuse.

Thanks,

Raymond Dalgleish.
Newsgroups: bionet.molbio.methds-reagnts
From: num208jn@MBDS.NRC.CA (John Nash)
Subject: Re: InVitroGen Dipsticks?
Reply-To: num208jn@MBDS.NRC.CA
Organization: NRC, OTTAWA, ONTARIO, CANADA.
Date: Wed, 19 Aug 92 19:07:06 GMT

In article <1992Aug19.171937.10562@magnus.acs.ohio-state.edu>, rrumpf@magnus.acs.ohio-state.edu (Robert Rumpf) writes:
>Any netheads out there have any experience with the InVitroGen Dipsticks used 
>for determining DNA quantity in solutions?  I have heard the literature claims 
>of being as easy to use as litmus paper, sensitive enough to detect 20ng/ul 
>with only a 1ul sample, etc.  What I would like are personal 
>experiences/opinions, etc on whether this stuff works, is cost-effective, 
>etc...
>
>Bob

The guy at the next workstation down from me tried them a coupla years
ago.  In my opinion, they were tedious, time-consuming and a pain to
use, and even though my colleague has left, the box has remained in my
fridge untouched. (If anybody passing through Ottawa wants it, drop
in).  

IMO, petri plates containing 20 ml of 1% agarose + 0.5 ug/ml ethidium
bromide viewed under UV, and photographed,  with your sample and some
lambda DNA standards (ranging from 0.5 ug/ml to 10 ug/ml - use 5 ul)
spotted on them and dried, are just as accurate, and since I pour
these plates once or twice a year and store them in the dark, less of
a pain to set up!!  (Ref: The Cold Spring Harbor Lab. Manual. of circa
1980).  These plates can detect at least 5 ul of 0.5 ng/ul DNA in my
(and everybody else I've talked to who's tried it's) hands.  I usually
dilute my sample to within the range of the standards, and use it to
assay DNA conc. before'n'after PCR, labelling, ligations, etc...

Caution:  they take a bit of experience to read as the DNA dries as
more as a ring than a disc... but so did the dipstick.

If Jim Graham were to hold a "useless kit of the year contest", this
kit would have my vote.


John Nash                            | Email: Nash@biologysx.lan.nrc.ca.
National Research Council of Canada  | Mail to "num208jn@mbds.nrc.ca"
Ottawa, Ontario, Canada              |    is forwarded automatically.

Disclaimer:  All opinions are mine, not NRC's!
*** "Dammit Jim!  I'm a Doctor not a tagline! *** (stolen from somebody)
Organization: Queen's University at Kingston
Date: Wednesday, 19 Aug 1992 08:00:48 EDT
From: <BACKD@QUCDN.QueensU.CA>
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Peltier PCR Instruments - Possible explaination of mixed reviews

I had the first MJ machine placed in Canada. The peltier units failed
about every 4 to 6 months. MJ was VERY accomodating in getting my unit
repaired and returned to me ASAP at no charge. They continued to do so
even after warrantee and even included software upgrades no charge.
The peltier problems were so bad that the sales rep at the FASEB booth
recognized my name and began a spontaneous round of apologies.

The last peltier was installed during the summer on 1990. Since then
we have had no further problems. I believe the MJ machine is one of
the most versatile machines on the market. We use it for PCR, sequencing
controlled rate annealling and occasionally for restriction digestion
run at odd temperatures.

Don Back
Department of Biochemistry
Queen's University
Kingston, Ontario. CANADA.
From: D31@EARN.TRANAVM1 (suleyman aydin)
Newsgroups: bionet.molbio.methds-reagnts
Subject: List on Chromatography. Exists any?
Date: 12 Aug 92 16:04:12 GMT
Distribution: bionet
Original-To: METHODS@UK.AC.DARESBURY


 
Dear Friends,
 
A friend of mine is asking me if a/any list(s) about Chromatography
exist(s) on the netland (internet, bitnet, uunet etc..)
 
She is a chemical engineer working on biological samples.
Any help is appreciated.
 
suleyman AYDIN
d31@tranavm1.bitnet
From: DMEIER@mis.mcw.edu
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: InVitroGen Dipsticks?
Date: 19 Aug 92 23:45:04 GMT
Distribution: bionet
Organization: Medical College of Wisconsin (Milwaukee, WI)

>Any netheads out there have any experience with the InVitroGen Dipsticks used
>for determining DNA quantity in solutions?  I have heard the literature
claims
>of being as easy to use as litmus paper, sensitive enough to detect 20ng/ul
>with only a 1ul sample, etc.  What I would like are personal
>experiences/opinions, etc on whether this stuff works, is cost-effective,
>etc...
>
>Bob

G'day all,
IMHO, these "Dipsticks" are not worth the money. They require you to
pipette on a small DNA sample to a plastic supported matrix, let it dry,
place the paper in a small cuvette with developer solution and let stand for
something like 10 minutes (I can't remember exactly as I only did it twice
...etc...
I agree completely with Bill.  It is difficult to "read" the color, partly 
because a darker ring forms with a pale interior.  In my opinion, the product 
has little value.

Dan Meier
From: Klaus.Matthaei@anu.edu.au
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Two Sequencing questions:
Date: 18 Aug 92 07:35:22 GMT
Distribution: bionet

>In a previous article, rrumpf@magnus.acs.ohio-state.edu (Robert Rumpf) says:
>
>>I have two questions regarding sequencing protocols:
>>
>>1.  Our 35-S gels are still sticky after drying; the saran wrap sticks to them 
>>and is difficult to remove.  Why?  We also noticed that removing the saran wrap
>>and placing film directly on the gels apparently causes some damage to the 
>>film from the moisture or urea in the gel...
>>
>>2.  Anyone have any experience with 33-P yet?  We would like to know if the 
>>energy from this isotope is sufficient to pass through saran wrap so we can 
>>expose films without taking the wrap off.
>>
>>Any help will be greatly appreciated...
>
>Don't know how much help I can offer but I can offer some sympathy.  I've
>recently tried 33P in dideoxy sequencing.  I don't "fix" the gel and I take
>the saran off after vacuum drying.  I did notice that the gel is definitely
>sticky but I was able to remove the wrap and the gel does not stick to the
>film.  I attributed the stickyness to the urea left in the gel.  I didn't
>usually observe much stickyness when I eluted the gel prior to drying while
>using 35S.  I'm running wedge gels and dry them at 80 deg C for one hour. 
>The stickyness is definitely worse at the thicker end.  I had thought that
>I might try drying for even longer but it hasn't been a problem for me yet
>and if I start drying for 2 hours I might just as well elute out the urea. 
>
>Please let me know if you try exposing with the film on or if someone else
>mailed you their experiences.
>-- 
>E-mail: mhollowa@ccmail.sunysb.edu (mail to freenet is forwarded)
>phone:  (516)444-3612
>I would claim to speak for my university but no one is likely to
>believe it.


We stopped using wedge sequencing gels years ago, mostly because they a
little harder to pour but also because of the drying problem.  We obtain
far better 'wedge' gels by adding a 1/3 volume of 1M Na acetate into the
bottom buffer tank just before loading the gel (after warming the gel). 
This gives beautiful compression of the bottom half of the gel and an
instant 'wedge'.

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
----------------------------------------------------------------------------
--

Newsgroups: bionet.molbio.methds-reagnts
From: Randy Hardy <hardy@mighty.facs.fccc.edu>
Subject: Re: Peltier PCR Instruments - Possible explaination of mixed reviews
Organization: I.C.R., Fox Chase Cancer Center
Date: Tue, 18 Aug 1992 18:59:16 GMT

In article <92231.105414REE700A@MAINE.MAINE.EDU> REE700A@MAINE.MAINE.EDU 
comments on the mixed reviews of Peltier element thermal cyclers and then notes
changes MJ research did to improve the reliability of the
commercially-available elements when used in cyclers.  He then ask:

>How many people that don't like Peltier
>systems have their adverse experience on POST-July '90 MJ RESEARCH
>instruments?

Well, we got an early MJ cycler several years ago which eventually developed
problems and was "upgraded" with a new element (under warranty),  Since then we
have had ZERO problems; this on a cycler that we use every day (often several
times/day).  A very satisfied user.

R. Hardy <hardy@mighty.facs.fccc.edu>
I.C.R., Fox Chase Cancer Center, 7701 Burholme Ave., Philadelphia, PA 19111,
USA
voice 215-728-2463   fax   215-728-2412

Newsgroups: bionet.molbio.methds-reagnts
From: ca566@cleveland.Freenet.Edu (Michael Holloway)
Subject: Re: Two Sequencing questions:
Reply-To: ca566@cleveland.Freenet.Edu (Michael Holloway)
Organization: Case Western Reserve University, Cleveland, OH (USA)
Date: Tue, 18 Aug 92 00:03:21 GMT


In a previous article, rrumpf@magnus.acs.ohio-state.edu (Robert Rumpf) says:

>I have two questions regarding sequencing protocols:
>
>1.  Our 35-S gels are still sticky after drying; the saran wrap sticks to them 
>and is difficult to remove.  Why?  We also noticed that removing the saran wrap
>and placing film directly on the gels apparently causes some damage to the 
>film from the moisture or urea in the gel...
>
>2.  Anyone have any experience with 33-P yet?  We would like to know if the 
>energy from this isotope is sufficient to pass through saran wrap so we can 
>expose films without taking the wrap off.
>
>Any help will be greatly appreciated...

Don't know how much help I can offer but I can offer some sympathy.  I've
recently tried 33P in dideoxy sequencing.  I don't "fix" the gel and I take
the saran off after vacuum drying.  I did notice that the gel is definitely
sticky but I was able to remove the wrap and the gel does not stick to the
film.  I attributed the stickyness to the urea left in the gel.  I didn't
usually observe much stickyness when I eluted the gel prior to drying while
using 35S.  I'm running wedge gels and dry them at 80 deg C for one hour. 
The stickyness is definitely worse at the thicker end.  I had thought that
I might try drying for even longer but it hasn't been a problem for me yet
and if I start drying for 2 hours I might just as well elute out the urea. 

Please let me know if you try exposing with the film on or if someone else
mailed you their experiences.
-- 
E-mail: mhollowa@ccmail.sunysb.edu (mail to freenet is forwarded)
phone:  (516)444-3612
I would claim to speak for my university but no one is likely to
believe it.
From: Klaus.Matthaei@anu.edu.au
Newsgroups: bionet.molbio.methds-reagnts
Subject: (none)
Date: 19 Aug 1992 01:52:41 GMT
Distribution: bionet

+  David L. Haviland, Ph.D.	     Internet:"haviland@kids.wustl.edu" 
Writes

"With interest I read your reply to question on sequencing gels.  You 
suggested adding 1/3 vol of NaAc (1M) to the bottom buffer just before 
running.  It sounds like an interesting idea and I intend to try it with my

next gel.  However, I have a question(s):  is it just a 1M solution of
NaAc, 
or is it made up to any particuar starting pH?  Is it made up in water, 
thus diluting the 1X TBE running buffer?

BTW - we don't dry our gels directly, the urea has caused us far too many 
problems, especially in the humid Mid-west.  We eliminated the Acetic Acid
fix, but we do soak and lift the gel off the plate in 10% EtOH, 90% water.
We let it soak there for about 20 mins, then we don't need to leave saran
wrap on for the exposure." 



I have had quite a bit of E-mail about my comment on the Na acetate method.
 I make the solution up in water and don't bother to Ph it.  Most comments
to me say that they also use the acetate but use a final concentration of
1M in the bottom tank.  I found my addition so good that I didn't bother to
try anything else.  It is possible of course that other concentrations are
better.  Maybe someone else out there has direct comparisons?

I also soak my gels for 20 minutes in 10% acetic,10% methanol before drying
for 1 hour.  I might add that we no longer run a lot of gels ourselves
since it is far cheaper if you don't have a lot of templates (and you have
to count  a technicians time) to use the ABI automated sequencer
(~$A20/template). (Unless of course you are a big cheese and have lots of
PhD students whose time costs nothing).

Cheers, Klaus

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

"Don't try and fix it if it ain't broke".
----------------------------------------------------------------------------
--
Newsgroups: bionet.molbio.methds-reagnts
From: John.Hardham@bbs.oit.unc.edu (John Hardham)
Subject: Re: MacVector
Organization: Extended Bulletin Board Service
Date: Fri, 14 Aug 1992 12:52:07 GMT

We have recently purchased MacMolly Tetra from ES2C and have been happy
with their package.  The program is actually set up in a modular format
and not an "all in one" format.  The Complign program can do sequence
alignments and multiple sequence alignments that can be exported to the
Compose program for a publication quality print out.
--
   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
From: browns@ccu.umanitoba.ca (Stuart Brown)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: HELP: ligation (used to be Re: cip & geneclean)
Date: 21 Aug 92 15:17:01 GMT
Distribution: bionet
Organization: University of Manitoba, Winnipeg, Manitoba, Canada

>>> 
>>>We have been attempting to clone a fragment of DNA into a high
>>>copy-number vector (a derivative of the pUC plasmids).  We have had some
>>>problems with incomplete digestion of the vector causing high
>>                ^^^^^^^^^^^^^^^^^^^^
>>>background, thus making identification of recombinant clones difficult
>>>(using lacZ blue/white identification).  
>> 
>Thank you (in advance) for any assistance with our problem,
>Vernon Coyne
>coyne@uctvax.uct.ac.za
>
>  

A simple method for reducing background from uncut vector: after digestion and
dephosphorylation, run your cut vector on a gel and cut out the band of linear
molecules.  Ligate in agarose or clean DNA out of agarose with your favorite
method.

	-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: mathog@seqvax.caltech.edu (David Mathog)
Subject: Sequencing survey
News-Software: VAX/VMS VNEWS 1.41    
Organization: Division of Biolgy, CALTECH
Date: Fri, 14 Aug 1992 01:22:00 GMT

We're thinking about setting up a sequencing facility for the division and are
now in the process of collecting feedback from people who have experience in
this area. If the shoe fits, please take a few minutes to fill out this
questionnaire, if not, no need to reply. 

Thanks in advance for your time,

David Mathog
mathog@seqvax.caltech.edu
manager, sequence analysis facility, biology division, Caltech




Background:

We currently have a central facility for analyzing sequence, but no
equivalent facility for generating it.  We would like to provide a
sequencing service that works something like this: 

 LAB ->  $$$ + DNA  -> Facility (shortest time possible) -> Sequence -+
  ^                                                                   |
  |                                                                   |
  +-------------------------------------------------------------------+

Each lab would do its own DNA preps, but all of the actual sequencing
reactions and reading of the sequences would be done by the central 
facility.  The final sequences would be deposited directly into the
end users' directories on our sequence analysis facility's computer.

A survey of the division indicated that roughly 2500 sequencing
(radioactive) gels are run each year. 



Questions:

1.  What has been your experience (positive and negative) with the 
various commercially available fluorescent sequencers?

2.  Which of the various types of fluorescent sequencing reactions 
have you tried and how well have they worked?

3a.  Assuming that the incoming DNA just needs to be pipetted into the
sequencing reactions how much manpower are we going to need to handle the
expected workload? 

3b.  How many machines will it require?

3c.  We'd like to turn around the average job in a day or less, is
this a reasonable goal?  What sort of schedule do you run?  How much 
do you charge/sequencing reaction (if you charge)?

4.  What software do you use to analyze the traces, how well does 
it work, and what does it cost?

5. If you've got the numbers - what does it REALLY cost you to
convert a single DNA sample to sequence?  If possible, break it down
reagents/overhead/labor/machinery/etc.  Also, if you've got the
numbers, what did it used to cost you to sequence with 35-S/32-P?  (We
will need at least an estimate of these numbers to justify the expense
of setting up the facility). 

6. If you ever used one of the commercial sequencing services how much
did it cost, what was their turn around time, and were you pleased
with the results? 

7.  If _you_ were going to set up a facility like this, how would you do 
it?

8.  Any other comments or suggestions?

Thank you for filling out the form!
From: rrumpf@magnus.acs.ohio-state.edu (Robert Rumpf)
Newsgroups: bionet.molbio.methds-reagnts
Subject: InVitroGen Dipsticks?
Date: 19 Aug 92 17:19:37 GMT
Organization: The Ohio State University

Any netheads out there have any experience with the InVitroGen Dipsticks used 
for determining DNA quantity in solutions?  I have heard the literature claims 
of being as easy to use as litmus paper, sensitive enough to detect 20ng/ul 
with only a 1ul sample, etc.  What I would like are personal 
experiences/opinions, etc on whether this stuff works, is cost-effective, 
etc...

Bob
From: brian@bio.aau.dk
Newsgroups: bionet.molbio.methds-reagnts
Subject: TMACl hybridization of RNA:DNA
Date: 14 Aug 92 15:20:15 GMT
Organization: BIOBASE, Denmark

Hello all you kind people out there!

I am trying to set up a system to catch some RNA in solution with at DNA oligo
of 22 bp. This oligo is attached to DYNA-beads.
So my question is: has anyone experience in this sort of hybridisation, 
especially I am interested in knowing if TMACl (tetramethylammonium chloride)
will have the same effect on RNA:DNA hybrides as on DNA:DNA hybrids. (I've
got some references on DNA:DNA, but haven't been able to find any on RNA:DNA).

Furthermore I would like if somebody has some experience on how to keep the
DYNA-beads from falling to the bottom (I know the hard way, resuspending them
mannully ones in a while, but as a normal human beeing, I am seeking for some-
thing to do the job for me)

Any suggestion is more than wellcome. 

Peter Kristensen
Department of chemistry
Division of Biostructural Chemistry
University of Aarhus
Denmark

e-mail : Brian@bio.aau.dk
From: kristoff@genbank.bio.net (David Kristofferson)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: acquisition of sequence from genbank
Date: 19 Aug 92 17:33:58 GMT
Organization: GenBank Online Service

ca566@cleveland.Freenet.Edu (Michael Holloway) writes:

>You can also telnet to genbank.bio.net or genbank.ig.com maintained by 

genbank.bio.net, not genbank.ig.com

>Intelligenetics.  They'll give you 10 or 15 minutes on-line.   
> login: genbank, password:4nigms	
>You'll be connected to an IRX interface that will search a number of
>sequence databases as well as the new data.

>Does anyone know what's going to happen to this service when Genbank moves?

You can do IRX-like searches by e-mail through NCBI's
retrieve@ncbi.nlm.nih.gov e-mail server.  Interactive access to IRX
will continue to be offerred by IntelliGenetics after the contract
ends on a commercial basis.

				Sincerely,

				Dave Kristofferson
				GenBank Manager

				kristoff@genbank.bio.net
From: afc@gnv.ifas.ufl.edu (Andrew F. Cockburn)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Superbroth and plasmid preps?
Date: 18 Aug 92 13:21:09 -0500
Organization: USDA/ARS/MAVERL

I saw an article a few weeks ago in a biotech company's newletter
touting the advantages of growing plasmids in rich media.  The 
newletter went the way of all such things, but now I wonder if I
should have kept it.

Does anyone have experience using rich media (eg 2x or 3x L broth)
for growing plasmids in E. coli?  Do the specific vector and host
matter?  Are the yields high enough to justify switching from L
broth?  What is your media recipe?  Do you use the same plasmid
prep as before?

Andrew Cockburn
USDA
From: penas@ucbeh.san.uc.edu
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re:  cDNA libraries (Reference)
Date: 14 Aug 92 14:30:33 EST
Distribution: world
Organization: Univ. of Cincinnati

Sorry I did not post this before.  The reference for the solution-phase
screening is:

Perez D.M., et.al.  Solution-phase screening ... Mol. Pharm. 1991; 40:876.


-- 
Sandra Pena de Ortiz     : penas@ucbeh.bitnet
University of Cincinnati
College of Medicine
Toxicology Program
From: William_D_WARREN@UMAIL.UMD.EDU (ww40)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: InVitroGen Dipsticks?
Date: 19 Aug 92 18:08:00 GMT
Distribution: bionet

>Any netheads out there have any experience with the InVitroGen Dipsticks used 
>for determining DNA quantity in solutions?  I have heard the literature
claims
>of being as easy to use as litmus paper, sensitive enough to detect 20ng/ul 
>with only a 1ul sample, etc.  What I would like are personal 
>experiences/opinions, etc on whether this stuff works, is cost-effective, 
>etc...
>
>Bob

G'day all,
IMHO, these "Dipsticks" are not worth the money. They require you to
pipette on a small DNA sample to a plastic supported matrix, let it dry,
place the paper in a small cuvette with developer solution and let stand for
something like 10 minutes (I can't remember exactly as I only did it twice
and decided that they weren't worth the effort), rinse the stick and place
it in a second solution for colour development (another 10 or so minutes)
before you get the result. Total time is around about 30minutes and even
then it's very difficult to determine the concentration accurately since
twice the DNA does not produce twice the colour at low concentration
(ie even if you set up a series of standards it's very difficult to tell the
difference between samples of 20ng and 40ng).

To anyone thinking of buying the DNA Dipstick I'd suggest that, unless
they have improved their product in the last year, you'd be better off
running your sample along with some standards on a mini agarose gel for
30min - in my hands this gives a more accurate estimation of DNA
concentration and also leaves you free for 30 minutes to do other things
(like read Bionet News :)

Bill Warren

Newsgroups: bionet.molbio.methds-reagnts