HDTenEightyP/NetNews huggingface dataset:
Organization: University of Maine System
Date: Tuesday, 28 Jul 1992 11:49:37 EDT
From: <IO00865@MAINE.MAINE.EDU>
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Need help on Northern hyb

You say that you "know that there is enough RNA on the membrane and that
it is expressed at moderate levels." How do you know these things?
DO you know that your RNA is not degraded? Do you know that RNA you have
loaded onto the gel is being transfered and retained by the membrane?
I would suggest that you find a good positive control. The best would
be to use RNA from a different source which has been shown to give
a signal on a Northern when probed for your gene. In addition probing
your blots with a probe which is present in high amounts will show
you if your blots are any good (How much RNA is there/ is it degraded).
Another possible problem is that your probe is no good. Make sure that
your oligo would detect the sense RNA (the oligo must be anti-sense).
and that your oligo is really labeled. Depending on how you purify
your oligo after labelling you may just be counting unicorporated
nucleotides.

If you want to send me more specifics on your methods I would be
willing to take a look at them...
Ethan Strauss                              IO00865@maine
Dept. Plant Biology & Pathology            Maple St. Apt.#1
University of Maine at Orono 04469         Bangor ME, 04401
207-581-2994                               207-947-6017
ps. I had a lot of trouble getting good transfer using cheap agarose.
If you are not already doing so try using FMC GTG grade agarose and see
if it helps.
Good Luck
From: kim@m44.unm.edu
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Cloning PCR products?
Date: 23 Jul 92 17:09:36 GMT
Reply-To: kim@m44.unm.edu
Organization: UNM Cancer Center, Albuquerque, NM

In article <1992Jul23.030739.10401@menudo.uh.edu>, bchs1b@Elroy.UH.EDU (Michael Benedik) writes:
>
>	There are a lot of different methods around for cloning PCR 
>products to get around the problems of the fragment ends left after 
>amplification. Does any one know whether this is solely a problem with
>TAQ polymerase? Are there other polymerases which leave nice ends
>suitable for subsequent cloning without going through the standard
>gyrations?


Nucleic Acids Research 19:184 (1991)
"Improved cloning efficiency of polymerase chain reaction (PCR) products after
proteinase K digestion."

Disclaimer:  I never tried this . . . I just collect them.
  This method is based on the hypothesis that the polymerase remains attached
to the end of the DNA, and is resistant to removal by standard methods.  They
do a proteinase K digestion after phenol and chloroform extraction and ethanol
precipitation.  then there was a second phenol chloroform extraction and
ethanol precipitation . . . what a lot of steps!  They show vastly improved
cloning efficiency.  I hope this is of some help.

Give us a follow-up if it works.

Daniel Kim
KIM@FLOVAX.LANL.GOV
From: rdna@matt.ksu.ksu.edu (Paul Paukstelis)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Buffer question
Date: 23 Jul 92 16:35:41 GMT
Organization: Kansas State University
NNTP-Posting-Host: matt.ksu.ksu.edu


A buffer question:

What is TBE, where can it be ordered, and are there any viable substitutes that
can be used?
Could EDTA possibly be one of these?

I'm a senior in high school doing a research project. Funds are short and TBE
is getting hard to find.

Ohh yeah, what precent solution should the TBE in.

Thanks a bundle in advance

rDNA of MLF
Paul Paukstelis
industriaL musiC froM manhattaN kS
rdna@matt.ksu.ksu.edu
Newsgroups: bionet.molbio.methds-reagnts
From: num208ww@MBDS.NRC.CA (Warren Wakarchuk)
Subject: Re: SS Phagemid gradients, KI or CsCl?
Reply-To: num208ww@MBDS.NRC.CA
Organization: NRC, OTTAWA, ONTARIO, CANADA.
Date: Thu, 23 Jul 92 03:10:26 GMT


In article <1992Jul21.154049.25431@usenet.ins.cwru.edu>, djt2@po.CWRU.Edu (Dennis J. Templeton) writes:
>
>We're trying to purify a mg or so of phagemid DNA, and need it to be free
>from contaminating double stranded DNA, such as might come from broken
>cells.  The protocol (sketchy) we're following suggests using EtBr/KI
>> stuff deleted
>dennis

Dennis, you might have better luck if you band the phage particles
themselves in CsCl, then simply extract DNA from the isolated phage
particles.  I used this technique about 10 years ago when I worked
with T7 phage particles.  I don't see why it wouldn't work with
phagemid.  Since the pariticles are lighter than DS DNA you should get
a good clean up.  I can't remember the exact reference, but I suspect
some of the earlier Methods in Enzymology (vol 65? 68?) may contain
references to banding phage in CsCl.  There is a series of books
published by the Cold Spring Habour Press on Lambda, T phage and SS
DNA phage, I am certain there will be references to how to do it. 
Good luck!
Dr. Warren Wakarchuk   Prefered email: wakarchu@biologysx.lan.nrc.ca
                       Alternate     : NUM208WW@MBDS.NRC.CA
IBS-Protein Structure and Design
National Research Council Canada
Ottawa, Ont.,
Canada K1A 0R6   ***THESE ARE MY OPINIONS NOT THOSE OF NRC***
Newsgroups: bionet.molbio.methds-reagnts
From: bchs1b@Elroy.UH.EDU (Michael Benedik)
Subject: Re: SS Phagemid gradients, KI or CsCl?
Reply-To: bchs1b@Elroy.UH.EDU
Organization: University of Houston
Date: Thu, 23 Jul 1992 02:56:22 GMT

In article <1992Jul21.154049.25431@usenet.ins.cwru.edu>, djt2@po.CWRU.Edu (Dennis J. Templeton) writes:
>
>We're trying to purify a mg or so of phagemid DNA, and need it to be free
>from contaminating double stranded DNA, such as might come from broken
>cells.  The protocol (sketchy) we're following suggests using EtBr/KI
>gradients for this, and collecting the lower band (SS) and discarding the
>upper band.  Our first go at this was a bust, as we lost almost all of the
>DNA in the work up. (the KI solution containing the DNA was sucked up into
>the H2O-saturated N Butanol!)
>
>I measured the density of the KI solution (1.6) and it is the same as our
>CsCl solution.  Obviously DS DNA floats in both near the middle of the
>tube.
>
>It made me ask myself, why KI and not CsCl?  Also, since we're pulling the
>lower band (we see only two, the lower is brighter) mightn't it be
>contaminated with some superhelical DNA along with the SS DNA?
>
>I really hate not knowing what's behind the reasoning for this protocol.
>
>Insights for why KI is used here and not CsCl would be appreciated, as
>would any favorite methods for purifying SS DNA away from DS DNA.  Some
>have suggested a low melt gel, but I'm hesitant, since we need a minimum of
>50 ug for each run.
>
>thanks 
>
>dennis

Instead of running gradients, why don't you treat the lysate with DNAse,
which will not get into the phage heads, then PEG ppt your phage (twice
if you want to be paranoid) phenol extract and etoh ppt. Should be
free of ds DNA.

	---------------------------------------------------
	 Michael Benedik
	 Department of Biochemical and Biophysical Sciences
	 University of Houston
	 INTERNET: Benedik@UH.EDU	BITNET: Benedik@UHOU
From: wetsel_r@wums.wustl.edu
Newsgroups: bionet.molbio.methds-reagnts
Subject: RE: S35- met labelling of proteins: URGENT
Date: 31 JUL 92 11:31:38 GMT
Organization: MIT PLASMA FUSION CENTER
NNTP-Posting-Host: msnews.wustl.edu

In a previous article, bchs1b@Elroy.UH.EDU (Michael Benedik) wrote:
>In article <30JUL92.14472467@wums.wustl.edu>, wetsel_r@wums.wustl.edu writes:
>>That's an open question, some additional details would help.  However, we
  ---- deleted ----->>
>>David
> 
>Is that a typo by chance, 0.250 uCi per ml. I would think that
>250 uCi or (.250 mCi) would be more appropriate, although I am not an
>expert at labelling tissue culture cells. We use 100 uCi per ml for
>bacteria.
>	---------------------------------------------------
>	 Michael Benedik
>	 Department of Biochemical and Biophysical Sciences
>	 University of Houston
>	 INTERNET: Benedik@UH.EDU	BITNET: Benedik@UHOU
------------

	Yes, yes, yes.... that was a typo and should be as you have stated. 
We use 250-500 uCi per ml for labeling.  My apologies to all involved!

	A second point that Martyn White (mkw6@dayhoff.med.virginia.edu) 
brought to my attention is that 35S-met is volitile (sp?) and *hot* by 
products can escape from your flask and stick to your incubator.  This 
speaks from experience from having to clean out the incubator after it 
"lights up" with 35S counts detected by wipe tests :-)   He went on to 
suggest that flasks and plates are best enclosed in some sort of baggie to 
limit the spread of 35S compounds.  Heed this warning lest you spend half a 
day using contrad to clean out your incubator... ::grin::

David
Organization: Penn State University
Date: Wed, 29 Jul 1992 15:14:35 EDT
From: <PXH@psuvm.psu.edu>
Newsgroups: bionet.molbio.methds-reagnts
Subject: help with det. micro. content of water

I am not sure this is the appropriate group for this post, but do bear with me!
I wonder if anyone out there could help me on this one.
There is a product marketed by a company called Medical Tech.
Corp. ( I think) which can be used for determining the
microbial contenet of water.  Basically, from what I
understand, it's like a slide covered with agar which you dip
in the water to be analysed.  You then incubate it
appropriately and can determine the contamination.  So here's
where I need help - are there any other
manufacturers/sources of this product?  Please e-mail me as I
am not a frequent reader of this group.  Thanks in advance!

Deepti Pradhan
Mon, 27 Jul 1992 14: 30:43 +0100
               Mon, 27 Jul 1992 14: 30:27 +0100
               Mon, 27 Jul 1992 12: 20:16 +0100
From: suter@de.dbp.MPG.MPIZ-KOELN.VAX
Newsgroups: bionet.molbio.methds-reagnts
Subject: urban "lab" legends
Date: 27 Jul 92 11:20:16 GMT
Distribution: bionet
Content-Identifier: 12
Original-To: methods@uk.ac.daresbury
Conversion: Prohibited

dear colleagues,

Ed Rybicki writes:

< Basavareju Shankarappa writes:
<>>>>I have observed that using NaCl for pptn of DNA often results in
<>>>>precipitation of salt specially after prolonged incubation at
<>>>>-20 C.  I learnt this from my experience rather than anything.
<> Disclaimer: Of course, my grad students put their DNA into the freezer
<> to precipitate...
<Why do they do that?  So ANNOYING!!  I used to think it was because older
<protocols had it in, but I now think it's because it's the lab equivalent of an
<urban legend, and the buggers don't ever bother to go and look up methods
<properly.
 
interesting point, about urban legends (lab legends ?). Here are a few more,
which are (in my humble opinion) either unnecessary or even counterproductive:
1. always store your 70% and 100% ethanol at -20C (main advantage: as
   soon as you open the bottle, water is sucked in, lowering your ethanol conc)
2. always hybridize in the presence of 0.1-0.5 % SDS (7% SDS is superior to
   suppress background, but we'll stick to the old protocol anyway)
3. in each ligation, use at least 1 micro liter of ligase (usually 100 fold 
   excess of enzyme, works better, eh?)
4. all southern filters should be baked at 80C...under vacuum (o.k. o.k.
   all manufacturers convince us this is only necessary for nitrocellulose,
   but....)
5. DNA should be dried in the speedvac for at least 10-20 minutes (why is
   it so hard to dissolve my pellet ?)
6. NaCl is the salt of choice for ethanol precipitations (NaAc ? NH4Ac ? 
   LiCl ? LiAc ? MgAc ? lets take a look in Sambrook !)
7. library screening. always use these round NC filters that fit exactly on 
   the agar plates...even when they are 10 times more expensive than the
   square ones you can cut from a roll !
8. And the last one: HeLa cells originate from Helen Lane or Henrietta Lacks
   (whereas insiders know they were invented by Henry "hip" LaBouchette !)

did i start some new rumours ?
   
cheers ! 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. xx49-221-5062.213
--------------------------------------------------------------------------------
Job offers will be rejected if a self-addressed, stamped envelope and $10
handling charge are not included.
-------------------------------> end of message <-------------------------------
From: BMGSLUIS@nl.LeidenUniv.rulgl
Newsgroups: bionet.molbio.methds-reagnts
Subject: Sources for proteinkinases sought
Date: 27 Jul 92 12:12:00 GMT
Distribution: bionet
X-Envelope-To: methods@daresbury.ac.uk
Original-To: methods@uk.ac.daresbury
X-Vms-To: IN%"methods@daresbury.ac.uk"

In our work on transcription, we would like to measure the effect of various
transcription factors in vitro. As phosphorylation is known to modulate
protein function and/or activity we would like to learn about sources or
distributors of proteinkinases.
Cees A. van Sluis
Molecular Genetics
Leiden University
PO Box 9502
NL-2300 RA Leiden, the Netherlands
Voice +31-71-274537
Voice +31-71-274758
Fax   +31-71-274537
From: mic268a@vaxc.cc.monash.edu.au
Newsgroups: bionet.molbio.methds-reagnts
Subject: Colony hyb with Bacillus
Date: 24 Jul 92 15:14:16 +1000
Organization: Computer Centre, Monash University, Australia

Can anyone out there please help with a method.
Does anyone know how to do a colony hybridisation on Bacillus subtilis?
Any help would be gratefully appreciated.

With many thanks
Marina Harper
Monash University
From: eric@bcserv.wustl.edu (Hugo (Eric R.))
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: What happens to 32P-labelled compounds?
Date: 31 Jul 92 10:43:44 GMT
Organization: Washington University in Saint Louis, MO USA
NNTP-Posting-Host: bcserv.wustl.edu

>In article <1992Jul30.085812.18345@polaris.utu.fi>
>eepee@polaris.utu.fi (Esa-Pekka P{lvim{ki) writes:

>>I understand that a S atom in ATP is replaced with a 32P atom to produce a
>>radioactive ATP molecule. When the 32P decays to 32S, the ATP molecule
>>continues to exist as a "real" ATP with a 32S where it should be.
>>I might be wrong, though.

>There is no S in ATP.  ATP = Adenosine triphosphate.  It contains 3 phosphate
>groups.  One of these is replaced by 32P to make the radioactive molecule.  Th
e
>chemistry of the ATP is (presumably almost) identical to that of one without
>the ATP.  One can label DNA or whatever with it.  When it decays, a near by
>film is exposed.  The molecule may or may not be destroyed (so this thread is
>an interesting one) but molecular biologists in general don't care because
>having seen that a decay occured, they don't care about the smoldering ruin (?
)
>left behind.  (That is, they are able to read the DNA sequence, so what if the
>actual DNA was destroyed in the process?)

>(You can also label ATP by replacing the P with 35S.)

>  Tom Schneider
>  National Cancer Institute
>  Laboratory of Mathematical Biology
>  Frederick, Maryland  21702-1201
>  toms@ncifcrf.gov


Actually its not the phosphorus atom that gets replaced in 35S labeled
nucleotides but rather the double-bonded O on the phosphate that is
substituted in thio nucleotides (they are still triphosphates).
-- 
  Eric R. Hugo, Postdoctoral Research Associate |eric@bcserv.wustl.edu
  Dept. of Biochemistry and Molecular Biophysics|                
  Washington University School of Medicine      |               
  Box 8231, St. Louis, MO 63110_________________| (314) 362-3342
From: mcgraw%gandal.dnet@SERVER.UGA.EDU
Newsgroups: bionet.molbio.methds-reagnts
Subject: RE: buffers
Date: 23 Jul 92 14:58:09 GMT
Distribution: bionet


Bill,

Your comments on pH and temperature with regard to Tris buffers make me 
wonder what happens to pH in a 10 mM Tris-buffered solution (say pH 8.,
for example) when you heat it to 55, 72, and 94 degrees.
Obviously, I'm talking about PCR.

Which leads to the more general question of to what extent does varying
pH affect PCR?  Any comments?

				Al McGraw
                                   University of Georgia
From: wmelchior@NTBTOX.NCTR.FDA.GOV ("Bill Melchior, NCTR/FDA")
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Acid Precipitation
Date: 23 Jul 92 13:12:41 GMT
Reply-To: wmelchior@NTDOC.NCTR.FDA.GOV
Distribution: bionet

>>When one filters the acid-precipitated material through a glass filter, does
>>the material actually _stick_ to the glass, or is this a simple particulate
>>filtration?  If the latter, Why do we use glass?  Is it just for acid
>>resistance?

It's probably just physically trapped.  I've not done it for many years, but
my recollection is that you want to use a weak vacuum, because a strong one
pulls the DNA through.

>>Can acid precipitated nucleic acids be pelleted and washed as a pellet?  How
>>hard do they have to be centrifuged for this?  Has anyone tried this before?  
>>I would like to try precipitating in 96-well dishes and spinning using a plate
>>carrier in my refrigerated centrifuge, but the maximum RCF is about 2500 x g. 
>>Does this sound sufficient?

>Yes, you certainly can precipitate nucleic acids with TCA in a centrifuge.
>Standard DNAse assays are based on the efficient removal of large DNA
>and the measuring of the acid soluble (small fragments) left in the sup.
>I am not sure if you have enough G force for the microtiter dishes or not,
>I guess it is worth a try. It may be quite dependent on the DNA concentration,
>in fact I would strongly guess that it is.

As MB says, centrifugation certainly works, though 2500 g sounds marginal.
Something else to check for is that TCA, an organic compound, doesn't
dissolve the plastic in your plates; it may be that your solution will be
dilute enough that it won't matter.
________________________________________________________________________________
The opinions stated are mine, not those of NCTR or its sponsoring organizations.

Bill Melchior                                ||   "You have lawyers the way
National Center for Toxicological Research   ||    other people have mice."
Jefferson, AR  72079                         ||
(501) 543-7206                               ||   -Kenneth Duncan, English
                                             ||    Health & Safety Executive,
WMELCHIOR@NTDOC.NCTR.FDA.GOV                 ||    to US regulators
________________________________________________________________________________
The opinions stated are mine, not those of NCTR or its sponsoring organizations.

Bill Melchior                                ||   "You have lawyers the way
National Center for Toxicological Research   ||    other people have mice."
Jefferson, AR  72079                         ||
(501) 543-7206                               ||   -Kenneth Duncan, English
                                             ||    Health & Safety Executive,
WMELCHIOR@NTDOC.NCTR.FDA.GOV                 ||    to US regulators
From: sbhattac@mrc-crc.ac.uk (Dr. Shoumo Bhattacharya B1 061)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Ecoli strains
Date: 23 Jul 92 11:11:33 GMT
Distribution: bionet

Can anyone tell me of a source where I can find E.coli strains and 
their genotypes?  Also are E. coli GM48 methylation defective (dam/dcm)? Many thanks  
Shoumo Bhattacharya
From: walterh@U.WASHINGTON.EDU (Walter Hill)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Colony hyb with Bacillus
Date: 24 Jul 92 14:09:25 GMT
Distribution: bionet

I presume that you are having difficulty with lysing the cells on the
solid support.  We've had good success with 0.5 M NaOH and 30 seconds
of microwaving at the "high" setting (600-700 Watts).  Alternatively,
you can wet the solid support with saline, microwave, and then denature
with 0.5N NaOH, neutralize, and use.
Good Luck, -Walt Hill

walterh@u.washington.edu (primary)           (secondary) WEH@FDACFSAN.BITNET
Food & Drug Admin., Seafood Products Research Center, Bothell, WA 98041-3012
VOICE:(206) 486-8788           "Cook it first"            (206) 483-4996:FAX 


On 24 Jul 1992 mic268a@vaxc.cc.monash.edu.au wrote:

> Can anyone out there please help with a method.
> Does anyone know how to do a colony hybridisation on Bacillus subtilis?
> Any help would be gratefully appreciated.
> 
> With many thanks
> Marina Harper
> Monash University
>
From: WMELCHIOR@NTET.NCTR.FDA.GOV (Bill Melchior, NCTR/FDA)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Design of expression plasmid
Date: 21 Jul 92 21:17:23 GMT
Distribution: bionet

I am considering putting a gene under lac control in pBR322, using the lac
promoter from pOP95-5 (F. Fuller, Gene 19, 43-54 (82)).  I have a question
(actually several, but I won't bother you with the others):  Fuller says that
the gene should be placed "near" the 3' end of the promoter, but he doesn't
say what "near" means, and I haven't been able to find out from any of the 
other sources I've consulted.  Does anyone know what the acceptable range
is for distances between, say, the "-10" region of the promoter and the
Shine-Dalgarno sequence or the initiation codon?  (My gene has a native S-D 
sequence and an initiation codon.  I'm not making a fusion protein.)  I 
don't need super efficient expression, but do need some.

Thanks, Bill
________________________________________________________________________________
The opinions stated are mine, not those of NCTR or its sponsoring organizations.

Bill Melchior                                ||   "You have lawyers the way
National Center for Toxicological Research   ||    other people have mice."
Jefferson, AR  72079                         ||
(501) 543-7206                               ||   -Kenneth Duncan, English
                                             ||    Health & Safety Executive,
WMELCHIOR@NTDOC.NCTR.FDA.GOV                 ||    to US regulators
Newsgroups: bionet.molbio.methds-reagnts
From: mhollowa@ccnova.sunysb.edu (Michael Holloway)
Subject: PC12 cell variation and differentiation
News-Software: VAX/VMS VNEWS 1.41    
Organization: State University of New York, Stony Brook
Date: Tue, 21 Jul 1992 23:08:00 GMT

Are there any folks out there in Netland working with PC12 cells?  I'm 
interested in hearing about your experiences.  Specifically, have you had 
trouble with variability of the cells with continuous passage?

Apparently there are major differences in media used and a suspicion that a 
large amount of differentiation has occurred between lab groups as the cells 
are being passaged.  The directions I've received from Simon Halegoua here at 
Stony Brook call for splitting 1:5 when the cells reach 6 to 7 X 10^6 in a 10cm 
dish.  When this is followed religously, the cells are supposed to be able to 
be passaged nearly continuously.  The practical problem I'm having as I start 
to culture them though is that getting a good cell count is impossible.  They 
do not come apart in trypsin and no amout of pipetting will separate them.  
Apparently, the way cell density is followed on a practical basis is to split 
the cells when a certain degree of confluence is reached.  The problem with 
this though is that the cells NEVER actually become confluent, they clump.  
I've learned then that the upshot is that successfully keeping PC12 cells going 
relies entirely on a qualitative "feel" for the look of the plate and the 
cells.  When they get "too clumpy" its time to go back to the frozens.

This is of particular importance to me now because of the results I've gotten 
in preliminary observations of PC12 cells differentiated -- not with NGF -- but 
with Na butyrate.  This is supposed to send the cells off toward a mature 
chromaffin cell morphology.  As I've tried to repeat observations I'm getting 
unexpected variability in the results which I attribute to the number of 
passages through which these cells have gone.

If anyone has any suggestions or similar experiences I'd be glad to hear from 
you. 

Mike
From: K_GARSON@UK.AC.ICRF
Newsgroups: bionet.molbio.methds-reagnts
Subject: SDS in Flakes
Date: 23 Jul 92 09:41:00 GMT
Distribution: bionet
Original-To: METHODS@UK.AC.DARESBURY