HDTenEightyP/NetNews huggingface dataset:
From: waite07@angis.su.oz.au ()
Subject: Re:Sodium phosphate buffer
Reply-To: waite07@angis.su.oz.au ()
Organization: ANGIS, The University of Sydney, Australia
Date: Wed, 22 Jul 1992 02:19:10 GMT



	When reference is made to sodium phosphate buffer of a particular pH,
what is meant, is a specific ratio of the mono- and di-sodium salts which gives
a correspondingly specific pH value. The ratios can be obtained from;

	Dawson, R. M. C., Elliot, D. C., Elliot, W. H. and Jones, K. M.,
	(1986). Data for biochemical research. 3rd ed. 432. Clarendon
	Press, Oxford.

	In case this reference can not be found, I include the relevant table;

	pH, 25 deg. C	x ml 0.2M Di-sodium	y ml 0.2M mono-sodium
	--------------------------------------------------------------
	5.8		4.0			46.0
	6.0		6.15			43.85
	6.2		9.25			40.75
	6.4		13.25			36.75
	6.6		18.75			31.25
	6.8		24.5			25.5
	7.0		30.5			19.5
	7.2		36.0			14.0
	7.4		40.5			9.5
	7.6		43.5			6.5
	7.8		45.75			4.25
	8.0		47.35			2.65
	--------------------------------------------------------------
	Original reference;
	 Gomori, after Sorensen, Meth. Enzymol. 1, 143 (1955)

	I usually assume that any dilution of a 0.2M solution will not change
the pH provided that nanopure, or equivalent, water is used.
	I hope this is of some help.

						Alex Jeffries


-------------------------------------------------------------------------------
Alex Jeffries
Plant Virology Lab.
Department of Plant Sciences		waite07@angis.su.oz.au (129.78.25.38)
Waite Agricultural Research Institute
The University of Adelaide		Tel.;  61  8  372 2392
South Australia
Australia
===============================================================================



Newsgroups: bionet.molbio.methds-reagnts,sci.chem,sci.misc
From: mkolberg@ulrik.uio.no (Morten Kolberg)
Subject: Re: What happens to 32P-labelled compounds?
Organization: University of Oslo
Date: Thu, 30 Jul 1992 12:51:08 GMT


>>
>>What happens to 32P-isotope labelled compounds when the 32P breaks down?
>>I am interested in 32P-gamma-adenosine triphosphate, for example, and
>>also oligomeric deoxynucleic acids labelled at their 5' termini with
>>32P by the enzyme polynucleotide kinase.
>>

[Stuff deleted]

>
>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.
>

Yes, indeed, you are... :-)

32P decays with a beta minus of a fairly high energy. As the particle "leaves"
the 32S nucleus will become a recoil energy, witch in turn may break the bonds.
If the bond breaks depends upon two factors:

- What the actual beta energy is. As the beta particles is emitted with a 
  energy between 0 and Emax KeV (Emax is for 32P 1700KeV)

- How the energy is transfered to the nucleus. As rotational, vibrational etc.

There is also a possibility of recombination as a broken bond results in a 
32S atom that may have a rather "strange" oxidaton number and electron 
configuration. That is: rather reactive.


With the high emax of 32P, the bonds may be broken fairly often. I do not know 
about the possiblity of recombination. But as the nucleic acid are fairly 
complex molecules (....) I would suppose that there is a large possibility that
they are destroied (more or less) during the disintegration of 32P. 
(Just my 0.05 Kr guess...) 

>/ep
>
>

Morten Kolberg
Studiing nuclear chemist
mkolberg@kelvin.uio.no
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: 3'RACE PCR
From: cytogen@chmeds.ac.nz (Martin Kennedy)
Date: 24 Jul 92 12:44:11 +1300
Distribution: world

In article <1992Jul20.215850.1540@ucbeh.san.uc.edu>, penas@ucbeh.san.uc.edu writes:
> Hi Netters!
> 
> I have been trying to perform 3'RACE PCR to isolate clones of about 1 kb.  I
> use 2-3 gene-specific nested primers and an anchore primer located at the end
> of the dT-tail.  So far I have not had any luck.  The products that I have
> cloned and sequence are artefacts containing the anhore primer at both ends.  
> > 
> 
> -- 
> Sandra Pena de Ortiz   


Sandra-

You may already be doing this, but I prime my cDNA with an oligodT primer
with a polylinker at its 5' end, and terminating at its 3' end with 
(A/C/G), which primes at the 5' end of the poly-A tail.  Then I do the
PCR with a gene specific primer and a primer specific for only the
polylinker sequence.

For the record, the primers I use are:

for cDNA synthesis:	TAG-EcoRI-XhoI-NotI-T(17)-(A/C/G)
anchor PCR primer:	TAG-EcoRI-XhoI-Not-I

The latter primer is 21 bases in length.  Maybe you ought to fiddle
with this to get the length and GC content the same as your gene-specific
primer.

This set up has worked on three different genes for me, but I do notice
the same sort of artifacts you are seeing in amongst the successful clones.
I tend to ligate the entire PCR rx into the vector, and maybe size selection
would remove some of these artifacts.

Cheers,

Martin.

Newsgroups: bionet.molbio.methds-reagnts
From: kim@m44.unm.edu
Subject: Acid Precipitation
Date: Wed, 22 Jul 92 17:53:08 GMT
Organization: UNM Cancer Center, Albuquerque, NM
Reply-To: kim@m44.unm.edu

Hello group:

Last year, I posted a question about TCA precipitation, and got several useful
replies.  . . .  I lost them :(

So here are a few questions.

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?

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?

Thanks for your patience.  

Daniel Kim
KIM@FLOVAX.LANL.GOV
From: walterh@U.WASHINGTON.EDU (Walter Hill)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: What happens to 32P-labelled compounds?
Date: 30 Jul 92 15:17:33 GMT
Distribution: bionet

At the risk of adding to the confusion regarding 32P and 32S, would the
decay of 32P result in a single stranded nick in a piece of double-stranded
DNA.  I seem to remember some ancient data (from about 25 years ago) using
phage T4 that concluded that one double stranded break was produced for every
10 single-stranded breaks.

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
From: rybicki@uctvax.uct.ac.za
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: ethanol precipitation
Date: 22 Jul 92 17:51:34 +0200
Distribution: bionet
Organization: University of Cape Town

In article <1992Jul17.111609.1306@gnv.ifas.ufl.edu>, afc@gnv.ifas.ufl.edu (Andrew F. Cockburn) writes:
> In article <9207152209.AA22355@phobos.med.pitt.edu>, bsh@MED.PITT.EDU (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. 
 _________________________________________________________________________
|                                 |                                       |
|  Ed Rybicki                     |    Now you've got the hang of it      |
|  Dept Microbiology              | There's nothing you can't do with it  |
|  University of Cape Town        |         If you're very into it        |
|  PB Rondebosch 7700             |         You can't go wrong....        |
|  South Africa                   |                                       |
|  fax 27 21 650 4023             |              - Mad John               |
|  ed@micro.uct.ac.za             | (Ogden's Nut-Gone Flake, Small Faces) |
|_________________________________|_______________________________________|
From: naffara@crc.ac.uk (Dr. N.A. Affara)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Help needed with strange PCR results!
Date: 21 Jul 92 17:17:15 GMT
Organization: MRC Human Genome Resource Centre

I have a problem with a PCR result.
The situation is this....
I am PCRing cDNA inserts cloned into lambda gt10 using primers at the edge 
of the vector arms. We are then sequencing the product.
One particular insert is getting on my nerves to say the least.
I have tried PCRing this on several occasions (using different reagents
in some cases) and I got a different combination of three bands.
These were 2.8kb, 1kb and 0.4kb. 
The last time I tried I got all three bands. Ah ha, I thought, now I'll be
clever, and cut out the top (2.8kb) band, geneclean it, and re-PCR it.
So, I went away and merrily did the above thinking I had finally outwitted
this lambda. 
But it got me back. When I looked at the gel I saw the bottom two bands but
none of the top one which I was supposed to be amplifying.

 ----- (2.8kb)

 -----  (1kb)

 
 ----- (0.4kb)

1st PCR                                      2nd re-PCR of top band

 ----- (only 2.8kb band)                                         (no top band)
       (cut out, geneclean,
        and re-PCR)                              ----- (1kb)


                                                 ----- (0.4kb) Only these two

What is happening???

The bands can't be concatomers (spelling?) because they don't multiply up
properly (unless the top band is two 1kb bands and two 0.4kb bands which I
think is highly unlikely.

It's too late to save me from going insane, but I would appreciate any 
help or comments.

Thanks,
Phil
E-mail naffara@uk.ac.crc
From: kumar@neb.com (Sanjay Kumar)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: The largest woman in the world (was: HeLa cells)
Date: 21 Jul 92 15:25:25 GMT
Distribution: bionet

For those interested in the origin of HeLa cells, a good 
book to read is "A Conspiracy of Cells" by Michael Gold.
It's light but interesting reading, and clearly states that
Henrietta Lacks is the source of HeLa cells.

Sanjay Kumar
kumar@neb.com

P.S.

Sorry if this is a duplicate posting.  We're in the middle of
an operating system upgrade and not everything is working just
right.
Newsgroups: bionet.molbio.methds-reagnts
From: robert@arbo.microbiol.uwa.oz.au (Robert Coelen)
Subject: re: Colony hyb with Bacillus
Organization: University of Western Australia
Date: Fri, 24 Jul 1992 08:13:51 GMT

In article <qzgmkh-@lynx.unm.edu> kim@m44.unm.edu writes:
>In article <1992Jul23.025622.9263@menudo.uh.edu>, bchs1b@Elroy.UH.EDU (Michael Benedik) writes:
>>In article <1992Jul21.154049.25431@usenet.ins.cwru.edu>, djt2@po.CWRU.Edu (Dennis J. Templeton) writes:
>
>
><Stuff about ss phagemid DNA preps omitted>
>
>Instead of using gradient centrifugation or PEG, there is a method that uses
>Glacial Acetic Acid and Sodium Perchlorate.  It is a glass-binding type DNA
>purification, like Geneclean.  I have never tried it, but the reference is:
>
>Nucleic Acids Research 15:5507-5516
>
>A modification of this method was posted a while ago as:
>
>1. In a 1.5 ml tube, mix 1 ml cleared phagemid supernatant with 10 microliters
>of glacial acetic acid.  Sit r.t. for 5 minutes to precipitate phage.
>
>2.  Spin out the phage pellet.  Drain.
>
>3.  Resuspend in 4.5 M Sodium Perchlorate (I didn't get the volume to use),
>made up in TE buffer.  Sit 50 C for 10 minutes.
>
>4.  Add 5 microliters glassmilk from Geneclean or home-made.  Rock on ice 10
>minutes.  (So the volume to resuspend should be maybe 100 - 300 microliters, I
>would guess).
>
>5.  Spin out the glass.  Drain.  Do NEW wash 3 times.
>
>6.  Thoroughly drain the glass pellet, and elute the ss DNA in 50 microliters
>water.
>
>
>If you try this, post a report on how it works/doesn't work.  I'd like to know.
>
>Daniel Kim
>KIM@FLOVAX.LANL.GOV


Lee Sammels in my lab cooked up a method that runs something like the
above and involves glass milk and the sequence we get from the ssDNA is
just BEAUTIFUL !!!!

Here are the salient details:

1.  Do a standard PEG/NaCl ppt of the phage from 1.5 ml of medium
2.  Discard supernatant
3.  Resuspend phage in 50 ul of 4.5M NaClO4 and combine 4 50 ul lots into
    one 200 ul lot
4.  Leave this with occasional vortexing at RT for 20'
5.  Add 10 ul of glass milk suspension and leave at RT for 15', vortex
    occasionally
6.  Spin the glass out, wash 3x with 50% etoh in TE
7.  Resuspend ssDNA in 20 ul of your favourite liquid
8.  Enjoy your sequence !!

This method is not yet optimised, but we'll get to that and repost. 


Robert Coelen
Dept of Microbiology
University of Western Australia
From: scottk@nuclease.berkeley.edu (Number Ten Ox)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: TouchDown PCR
Date: 30 Jul 92 10:49:49 GMT
Distribution: world
Organization: Biochemistry and Molecular Biology, U.C. Berkeley
NNTP-Posting-Host: nuclease.berkeley.edu

In article <36279@sdcc12.ucsd.edu>, rounsley@jeeves.ucsd.edu 
(Steve Rounsley) writes:
|> Quick question...
|> 	does anybody know what conditions to use for "Touchdown"
|> PCR.  I came across this term, and I don't know what kind of
|> cycles to use.
|> 	thanks in advance
|> 		Steve Rounsley 


The exact conditions will depend on what you're trying to do. The
reference for touchdown PCR is:

Don, et al. (1991) NAR 19(14): 4008

Basically, touchdown PCR involves decreasing the annealing temp
by 1 degree C every second cycle to a 'touchdown' annealing temp
which is then used for 10 or so cycles.  It was originally intended
to bypass more complicated optimization processes for determining
optimal annealing temps. The idea is that any differences in Tm
between correct and incorrect annealing gives a 2-fold difference
in product amount per cycle (4-fold per degree C). You therefore
enrich for the correct product over any incorrect products.

Another use for this procedure is in determining DNA sequence for a
known peptide sequence. The strategy here is to use two sets of
degenerate primers that match potential coding sequences at the
two ends of a peptide of known sequence. In practice, this requires
that you know a stretch of peptide sequence of only 13 amino acids,
with left and right primers of 18 nt (6 a.a.) and a space in between
of one or more nt. Using these degenerate primers, you do a touchdown
PCR. You will get a huge number of products, but you can select the
desired product based on size since you know the exact interprimer
distance from the peptide sequence. The advantage of this technique is
that the touchdown PCR enriches for products containing correct matches
between primers and template. If you clone and sequence a dozen
PCR products, you can determine the correct coding sequence for
the peptide, design an oligo for hybridization, and ZOOOM...
cloned gene is practically in hand. The technique is obviously
especially useful for peptide sequences full of ser, lys, and arg
(six codons each).

I haven't seen this latter use of touchdown PCR published. I got
it from folks in the Tjian lab here at Berkeley, but I have no
idea whether they made it up or heard it from someone else.


-- 
       ____         Scott Keeney, DNA repair-queer         ____
       \  /           scottk@mendel.berkeley.edu           \  /
        \/        Biochemistry and Molecular Biology        \/ 
                            U.C. Berkeley
From: ray@uk.ac.le ("Dr R. Dalgleish")
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Smiles on Southerns
Date: 30 Jul 92 12:40:52 GMT
Distribution: bionet
Original-To: methods@uk.ac.daresbury
X-Mailer: ELM [version 2.3 PL0 (LUT)]

If you run a large amount of DNA in a lane of a gel containing ethidium bromide
the DNA tends to bind the dye. If the ethidium bromide is limiting then there
may not be sufficient to saturate the DNA. At the _edges_ of the lane there
will be more ethidium bromide available (it will be able to diffuse in from the
spacer agarose between lanes) and so the DNA at the edges will tend to have
more ethidium bromide bound. The efect of this will be to retard the DNA at the
lane edges, hence smiling.

This is easily tested. If you load decreasing amounts of DNA the smiling will
reduce as the amount of ethidium bromide available will not be limiting for
lesser amounts of DNA. Increasing the ethidium bromide concentration will also
counter the effect.

If you still need convincing then set up two gels that differ only by one
containing ethidium bromide and the other not. Run identical samples of DNA
(using amounts known to normally induce smiling) on both gels for the same
amount of time. After electrophoresis stain the gel which had not contained
ethidium bromide. Look, no smiles!

Hope that this helps.


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  Department of Genetics,  EARN/BITNET: ray%leicester.ac.uk@ukacrl
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  University Road,                UUCP: ray%leicester.ac.uk@ukc.uucp
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From: skspoidn@uk.ac.rdg.susssys1 (Mike Poidinger)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Sodium Phosphates
Date: 21 Jul 92 10:18:44 GMT
Distribution: bionet
Original-To: methods@uk.ac.daresbury

When making 20x SSPE, you will in the end be adjusting 
the pH with NaOH, thus creating the correct 
disodium/monosodium ratio regardless of starting materials.

Assuming your solution starts acidic, of course. 
ie use the monosodium salt (most acidic), and adjust pH from there.

Mike Poidinger
Dept of Microbiology
University of Reading
From: AWS4270%USA.decnet@USAV01.GLAXO.COM ("USA::AWS4270")
Newsgroups: bionet.molbio.methds-reagnts
Subject: Polymerase fidelity
Date: 31 Jul 92 19:44:00 GMT
Distribution: bionet

With respect to fidelity in PCR reactions, which of the following
polymerases are recommended for best results:

1. Vent

2. Deep Vent

3.  Pfu

I'd appreciate any feedback; I don't know which one to order...
what pros and cons are associated with each, etc..

Thanks,

Anne
From: robert@arbo.microbiol.uwa.oz.au (Robert Coelen)
Subject: re: two first PCR cycles with long times?
Organization: University of Western Australia
Date: Mon, 27 Jul 1992 03:01:34 GMT

In article <1992Jul24.220831.17026@ulrik.uio.no> MREEBEN@BIOMED.UIO.NO (Mati Reeben) writes:
>
>Accidentally in a recent research article in Science from 3 July 1992 Vol. 257
>p.55 I came over following PCR conditions. 
>
>....All PCR were performed on single stranded cDNA's generated from
>poly(A)+-enriched RNA from different brain regions.
>The PCR conditions with nonredundant oligonucleotides consisted of two cycles
>at 95 C (5 minutes), 62 to 68 C (2 minutes), and 72 C (10 minutes) followed by
>25 cycles of 95 C (1 min), 62 to 68 C (2 minutes), and 72 C (2 minutes).
>
>I would be grateful, if somebody can explain me the sense of these first two
>cycles. OK, I understand initial more thorough denaturation, but why second
>long denaturation ? And why two 10 min extensions in the beginning ? Is initial
>template considered to be worse for elongation ? And how much this trick can
>improve PCR ?
>
It seems to me that the second long cycle is a bit of insurance in case the first
one did not snag the required product.


Robert Coelen 
Microbiology
UWA  
Newsgroups: bionet.molbio.methds-reagnts
From: bchs1b@Elroy.UH.EDU (Michael Benedik)
Subject: Re: Mutation Selection
Reply-To: bchs1b@Elroy.UH.EDU
Organization: University of Houston
Date: Fri, 31 Jul 1992 04:25:33 GMT

In article <1992Jul30.144803.34203@urz.unibas.ch>, herzer@urz.unibas.ch writes:
>I would like to look at the mutation rate of certain strains of E. coli
>and am looking for genes within E. coli for which mutations could be
>screened for efficiently.  For example, a gene which aquires a mutation
>such that it does grow on a certain plate whereas the wild-type would
>die; or color difference, etc.  Any help would be most appreciated.
>
>                                  Sincerely,
>                                             Peter
>                                             "Herzer@urz.unibas.ch"
>
There are a large number of these, for example resistance to 
nalidixic acid, rifampicin, valine and numerouse others. There is a chapter
on selectable phenotypes in the E. coli and Salmonella book 
(from ASM, edited by Neidhardt et al) in volume 2 which describes a large
number of such selectable markers and gives the references. But th e
bottom line is that there are hundreds (at least), although some do
depend on starting genotype.

	---------------------------------------------------
	 Michael Benedik
	 Department of Biochemical and Biophysical Sciences
	 University of Houston
	 INTERNET: Benedik@UH.EDU	BITNET: Benedik@UHOU
Newsgroups: bionet.molbio.methds-reagnts
From: Ola Myklebost
Subject: Re: Tracer Dyes in Agarose
Organization: The Norwegian Radium Hospital ICR
Date: Fri, 31 Jul 1992 10:30:43 GMT

In article <1992Jul31.102351.10004@ulrik.uio.no> Ola Myklebost writes:
>In article <3643@fcs280s.ncifcrf.gov> pnh@fcsparc6.ncifcrf.gov (Paul N Hengen)
>writes:
>..
>..
>>I'm wondering if anyone uses a dye that migrates faster than the
>>bromphenol blue front?
>
>We use Xylene Xyanole FF (Sigma X-0377)
>
>SORRY:
We use Xylenol Orange Sigma X-3500, that moves in the buffer front.
Newsgroups: bionet.molbio.methds-reagnts
From: djt2@po.CWRU.Edu (Dennis J. Templeton)
Subject: Re: Tracer Dyes in Agarose
Reply-To: djt2@po.CWRU.Edu (Dennis J. Templeton)
Organization: Case Western Reserve University, Cleveland, OH (USA)
Date: Mon, 27 Jul 92 21:35:42 GMT


In a previous article, pnh@fcsparc6.ncifcrf.gov (Paul N Hengen) says:

>I'm wondering if anyone uses a dye that migrates faster than the
>bromphenol blue front?
>

We use Orange G sometimes that migrates ahead of the BPB. The sole advantage, 
as I see it is that it doesn't block the DNA fluorescence.

good luck, dennis
Newsgroups: bionet.molbio.methds-reagnts