HDTenEightyP/NetNews huggingface dataset:
From: skspoidn@susssys1.rdg.ac.uk (Mike Poidinger)
Newsgroups: bionet.molbio.methds-reagnts
Subject: re: Genomic DNA prep
Date: 10 Apr 92 13:01:22 GMT
Distribution: bionet


re:

----- Begin Included Message -----


Message-Id: <9204091704.AA23462@genbank.bio.net>
To: methods-and-reagents@net.bio.genbank
From: kseiden@edu.nwu.acns.casbah (Kenneth Seidenman)
Subject: Re: Preparation of Genomic DNA
Date: 9 Apr 92 16:31:33 GMT

Hi,

I was wondering if anyone had a good protocol for preparing genomic DNA from
frozen liver tissue.  Since this DNA will be used for PCR, I'm particularly
concerned about cross-contamination between samples during the "pulverization"
steps.  I tried using a hammer and some tin foil, but I found this to be
rather messy and think there's a fair chance of cross-contamination.  Any
suggestions would be appreciated.

Thanks

Ken


----- End Included Message -----


I have prepared DNA for PCR and subsequent sequencing using a
(standard) protocol involving grinding tissue in a mortar and pestle
followed by SDS/prot K/salt incubation and phenol extraction
(I will send the full protocol if you want it)

As far as cross contamination goes, I have avoided that by using
a different motar+pestle for each sample, or by _thorough cleaning of
the instruments between uses. The lab I used to work in ground many
mosquito samples for virus isolation, and solved contamination
problems by having >50 glass grinders, cheap to buy, easy to use,
reasonably easy to clean afterwards.

-hope this helps, or am I missing the point?

Mike Poidinger
Dept of Microbiology
University of Reading
From: danj@WELCHGATE.WELCH.JHU.EDU (Dan Jacobson)
Newsgroups: bionet.molbio.methds-reagnts
Subject: *EcoP1
Date: 14 Apr 92 20:56:03 GMT
Distribution: bionet

Paul Fisher writes:

>We are looking for a source for a restriction
>enzyme called EcoP1. It is not listed in the most recent
>restriction databases although the GCG programme map
>knew about it one year ago. Apparently GCG has now
>forgotten what this enzyme is. Does it still exist?
>Where can we get it from? Thanks for your help.
>P.S. We would also like to know what the recognition site is.

REBASE (via WAIS) gives the following information about EcoP1:



<1>EcoPI
<2>
<3>Escherichia coli (PI)
<4>K. Murray
<5>AGACC
<6>3(6)
<7>
<8>Bachi, B., Reiser, J., Pirrotta, V. Methylation and cleavage sequences of the EcoP1 restriction-modification enzyme.  J. Mol. Biol. 128: 143-163 (1979)

Brockes, J.P. The deoxyribonucleic acid-modification enzyme of bacteriophage P1. Subunit structure.  Biochem. J. 133: 629-633 (1973)

Brockes, J.P., Brown, P.R., Murray, K. The deoxyribonucleic acid modification enzyme of bacteriophage P1: Purification and properties  Biochem. J. 127: 1-10 (1972)

Haberman, A. The bacteriophage P1 restriction endonuclease.  J. Mol. Biol. 89: 545-563 (1974)

Hattman, S., Brooks, J.E., Masurekar, M. Sequence specificity of the P1 modification methylase (M.EcoP1) and the DNA methylase (M.Ecodam) controlled by the Escherichia coli dam gene.  J. Mol. Biol. 126: 367-380 (1978)

You may want to get in touch with one of the groups in these papers to
find out more about a source for this enzyme.

Hope this helps,

Dan Jacobson

danj@welchgate.welch.jhu.edu
From: MPB@CBR.DWE.CSIRO.AU (Mark Bradley)
Newsgroups: bionet.molbio.methds-reagnts
Subject: acrylamide
Date: 16 Apr 92 13:32:53 GMT
Distribution: bionet

We have recently found that it is impossible to dissolve acrylamide
in very high quality miillipore QV grade water. On the other
hand it dissolves well on RO water. Obviously a few "ions"
need to be present.
From: Jennifer.Loros@dartmouth.edu (Jennifer Loros)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: RNase free DNase.
Date: 16 Apr 92 18:43:08 GMT
Article-I.D.: dartvax.1992Apr16.184308.13318
 <k?o.702931372@soma.umdnj.edu>
Organization: Dartmouth College, Hanover, NH

Hi Bruce,

We have had good success with Worthington Biochemicals and
Collaborative Research.

Good luck.

Jennifer Loros
From: saparici@crc.ac.uk (Dr. S.A.J.R. Aparicio)
Newsgroups: bionet.molbio.methds-reagnts
Subject: RNAase free DNAase
Date: 9 Apr 92 15:48:41 GMT
Reply-To: saparici@crc.ac.uk (Dr. S.A.J.R. Aparicio)
Organization: MRC Human Genome Mapping Project Resource Centre, Harrow, UK

We buy our DNAse from Sigma and find that it is quite RNAse free. 



(usual disclaimers)


Sam Aparicio
From: toms@fcs260c2.ncifcrf.gov (Tom Schneider)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Breaking the biotin/streptavidin bond
Date: 10 Apr 92 16:45:29 GMT
Organization: NCI Supercomputer Facility, Frederick, MD

Biotin binds to streptavidin quite strongly, which is what makes it a nice
system for binding DNA to magnetic beads.  But what if one wants to remove the
DNA from the beads later?  Does anyone know a method?  Clearly one way might be
to use a protease, but suppose we don't want to add any proteins to the DNA,
what could be done then?  Also, what happens if the beads with attached
streptavadin-biotin-DNA are boiled?

  Tom Schneider
  National Cancer Institute
  Laboratory of Mathematical Biology
  Frederick, Maryland  21702-1201
  toms@ncifcrf.gov
From: DELANEY@SALK-SC2.SDSC.EDU (Terry Delaney)
Newsgroups: bionet.molbio.methds-reagnts
Subject: subcloning YACs...
Date: 11 Apr 92 00:34:34 GMT
Distribution: bionet

Gentle readers,
I have been walking to an interesting Arabidopsis gene, and am attempting
to subclone a YAC into plasmid and cosmid or lambda vectors.  The first 
attempts at accomplishing this seemingly trivial task have been unsuccessful.
If any of the readers of this column can be of assistance, I would be 
grateful to hear from you.  Replies can be either to this service, or to me
personally.  In the latter case, I can share with the network any information 
I collect, if it seems helpful.  If any reader knows of a network directed 
toward genome project or YAC technology related issues, perhaps you could 
share that information too.

Thanks 10^6,
Terry Delaney
Salk Institute
From: browns@ccu.umanitoba.ca (Stuart Brown)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Is there a used lab equipment board here?
Date: 8 Apr 92 21:18:22 GMT
Organization: University of Manitoba, Winnipeg, Manitoba, Canada

In article <1992Apr3.223306.10357@usenet.ins.cwru.edu> djt2@po.CWRU.Edu (Dennis J. Templeton) writes:
>
>Dear powers-that-be;
>
>The net seems to be a perfect place to establish a used lab equipment         
>exchange bulletin board.  Is there one in existence here? and just where,     
>pray tell?
>
>Really, this would be very useful to those of us on a limited budget,       
>reduce waste, clear out storerooms, etc.  If one does NOT exist, maybe one    
>should be established.
>
>thanks...
>
>dennis

My wife is currently running a used equipment project at Cornell designed to 
move stuff from the University into High School classrooms (contact her:
Kim_Foglia@qmrelay.mail.cornell.edu).  Even very obsolete stuff can be 
valuable to a High School teacher.
	This net might be useful for such a thing in other localities (they
use a modified LAN at Cornell where the teachers can call into a central 
file server by modem without full network access).
	-Stu

-- 
Stuart M. Brown                               Postdocs rush in where wise men
U. of Manitoba, Dept. Plant Science, Canada       Fear to tread. 
browns@ccu.umanitoba.ca        
				Disclaimer?? Sure: "That Disc is Mine!"
From: kim@m44.unm.edu
Newsgroups: bionet.molbio.methds-reagnts
Subject: RNA Mol.Weight Markers
Date: 15 Apr 92 23:02:30 GMT
Article-I.D.: lynx.9dzjqdn
Reply-To: kim@m44.unm.edu
Organization: UNM Cancer Center, Albuquerque, NM

Hello group:

I am going to be doing Northern blots, and I will be needing mol. weight
markers.  I had read that DNA and RNA migration rates are equivalent in
glyoxal/DMSO gels, and so DNA markers can be used as standards in RNA gels of
this type.  Does anyone know whether this is true of Formaldehyde/Formamide
gels?

Also, in case I decide to go with glyoxal/DMSO, does anyone have a favorite
source of good deionized glyoxal?  The only stuff I have on hand here is very
old and has bits of stuff floating in it.  I'd rather not risk using it if it
can be avoided.  When I try to deionize it myself using AG501-X8, I seem to
have no success, although pH paper maybe is not a good way to test the quality
of glyoxal deionization.

Thanks.

Danny Kim
From: verwaerde@mbcl.rutgers.edu
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Thesit
Date: 16 Apr 92 19:56:52 GMT
Distribution: bionet

In article <9204091426.AA01788@server.uga.edu>, mcgraw%gandal.dnet@SERVER.UGA.EDU writes:
> Hello Netters,
> 
> A recent short article in NAR (Ponce, M.R., and Nicol, J.L., 1992, Vol. 20, 
> p.623) describes conditions for PCR amplification of long (>6 kb) DNA
> fragments.  The 10X buffer contains 1% Thesit. The SIGMA catalog says
> Thesit is a trade name for polyoxyethylene 9-lauryl ether.
Thesit ?

It sounds like a specific scientist disease...  :-))))))

> 
> Can anyone out there tell us more about Thesit in PCR?  If it's good, why
> is it good?  Where did you get it?  Any comments appreciated.
> 
> 				Thanks, 
> 
> 				Al McGraw
> 				University of Georgia
Newsgroups: bionet.molbio.methds-reagnts
From: gchacko@magnus.acs.ohio-state.edu (George W Chacko)
Subject: Re: RT-Inverse PCR
Organization: The Ohio State University
Date: Thu, 16 Apr 1992 22:19:03 GMT


Has anyone out there ever performed inverse PCR on RT products. If so, is 
there a single stranded ligase that one can use or do you have to synthesis a
second cDNA strand and then ligate to circularise?

Thanks

George
From: micprf@lure.latrobe.edu.au
Newsgroups: bionet.molbio.methds-reagnts
Subject: EcoP1 where are you?
Date: 14 Apr 92 13:56:40 GMT
Organization: VAX Cluster, Computer Centre, La Trobe University

Dear molbio freaks,
	We are looking for a source for a restriction
enzyme called EcoP1. It is not listed in the most recent
restriction databases although the GCG programme map
knew about it one year ago. Apparently GCG has now
forgotten what this enzyme is. Does it still exist?
Where can we get it from? Thanks for your help.
P.S. We would also like to know what the recognition site is.
Please direct answers by email to fisher@mpib-martinsried.mpg.dbp.de
Cheers, Paul Fisher.
Newsgroups: bionet.molbio.methds-reagnts
From: bchs1b@Elroy.UH.EDU (Michael Benedik)
Subject: Re: protease mutants in fungi
Reply-To: bchs1b@Elroy.UH.EDU
Organization: University of Houston
Distribution: bionet
Date: Thu, 9 Apr 1992 16:54:30 GMT

In article <07APR92.17800955.0011.MUSIC@SEMOVM>, C749SCB@SEMOVM.SEMO.EDU (Walt Lilly) writes:
>We are trying to isolate mutants of a wood-decaying fungus that are
>deficient in specific proteolytic enzyme activities.  Can anyone
>offer advice on a selection scheme (using liquid media or plates)
>which might allow us to find such mutants?  We know that the proposition
>will be complicated by the fact that the fungus produces and secretes
>several different proteases, including both endo- and exo- peptidases.
>
>Does anyone know of any commercially available peptides conjugated to
>a toxin....this seems like it might be an approach to select only for
>protease- mutants.  Any thoughts?
>
>************************************************************************
>*  WALT LILLY                     *                                    *
>*  DEPARTMENT OF BIOLOGY          *  INTERNET: C749SCB@SEMOVM.SEMO.EDU *
>*  SOUTHEAST MISSOURI STATE UNIV. *  BITNET: C749SCB@SEMOVM            *
>*  CAPE GIRARDEAU, MO 63701       *                                    *
>************************************************************************

I have screened for protease mutants in a number of different bacteria
using different medias. For most general proteases you can use
milk plates, you incorporate non-fat powdered milk into nutrient agar
plates (or appropriate rich growth plates). Final concentration of milk
about 1%. We usually prepare a 10% solution of milk and autoclave
separately for a short time (5 minutes) then pour into somewhat cooled media
Otherwise the milk curdles if too hot for too long.
Other alternative medias are gelatin and casein plates. We find that
different proteases give different signals on the different plates so
you can play around a little. You might try running zymograms off 
native gels to see which proteases give signals on which media.


	---------------------------------------------------
	 Michael Benedik
	 Department of Biochemical and Biophysical Sciences
	 University of Houston
	 INTERNET: Benedik@UH.EDU	BITNET: Benedik@UHOU
From: tewari@ccu.umanitoba.ca (Sandhya Tewari)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Biotin-11-dUTP supplier
Date: 15 Apr 92 15:24:58 GMT
Distribution: bionet.molbio.methds-reagents
Organization: University of Manitoba, Winnipeg, Manitoba, Canada

Hi netters
I am using a non-radioactive detection system using biotin-11-dUTP.
Those of you who are  using bio-11-dUTP too, could you please let
me know where you can order bio--11-dUTP without ordering a kit
along with it?  I tried Sigma but they put it on backorder and I
kept waiting almost for four months till they have finally deleted
the item from their catalog. They are supposedly supplying it under
a different cat. # now but I'm not prepared to wait anymore. Is 
there a reliable supplier?
Thank you in anticipation.

Sandhya Tewari
University of Manitoba
Newsgroups: bionet.molbio.methds-reagnts
From: bchs1b@Elroy.UH.EDU (Michael Benedik)
Subject: Re: Expression without fusion
Reply-To: bchs1b@Elroy.UH.EDU
Organization: University of Houston
Date: Thu, 16 Apr 1992 13:31:14 GMT

In article <1196.29ec0036@mbcl.rutgers.edu>, sofer@mbcl.rutgers.edu writes:
>Does anyone know of a lambda phage expression
>vector that does -not- need require that a fusion
>protein in order for one to detect recombinants.be made 
>
>I'm looking for an insertion vector rather than
>a replacement vector.
>
>Bill Sofer
>sofer@mbcl.rutgers.edu

It is not clear exactly what you are trying to do, but even the "fusion"
vectors do not require that a gene be fused to be expressed. If you clone
an intact expressable gene in lambda gt11 you can get expression not as
a fusion if your insert has the appropriate translational signals. There
are a variety of other insertion vectors which are also useable depending
upon what your final goals are.

	---------------------------------------------------
	 Michael Benedik
	 Department of Biochemical and Biophysical Sciences
	 University of Houston
	 INTERNET: Benedik@UH.EDU	BITNET: Benedik@UHOU
From: NAGEL@EGFRCUVX.BITNET ("NAT. AGRIC. GENETIC. ENG. LAB")
Newsgroups: bionet.molbio.methds-reagnts
Subject: paper supply
Date: 12 Apr 92 17:39:00 GMT
Article-I.D.: genbank.9204121543.AA23729
Distribution: bionet

Dear Netters,
              Can any one supply me with the following paper:
The sulfer-rich 2S Brazil nut albumin, published in Plant Mol.Bio
8,239-250 (1987) Altenbach et.al. (1987).
My E-mail address is: nagel%egfrcuvx.bitnet@ibm.gwdg.de

                                       Dr. Mouhmed Soliman.
From: braam@RICE.EDU (Janet Braam)
Newsgroups: bionet.molbio.methds-reagnts
Subject: (none)
Date: 14 Apr 92 14:39:06 GMT
Article-I.D.: bioc.388.braam
Reply-To: braam@rice.edu
Distribution: bionet

In article <12063.9204130857@subnode.susssys1.rdg.ac.uk>, skspoidn@susssys1.rdg.
ac.uk (Mike Poidinger) writes...
>Help.
> 
>I am trying to redissolve DNA which has been glass-rod-extracted
>according to protocol 3 from Sambrook/Maniatis, and the %$#@ material
>wont dissolve, wont even come off the glass rod. I am trying to prepare
>medium sized genomic DNA from cell monolayers for southern Hybridization.
> 
>Any ideas on
>a) Getting the DNA off the glass or
>b) A better procedure for DNA preparation
> 
>would be greatly appreciated
> 
>Thanks in advance,
> 
>Mike Poidinger
>Dept of Microbiology
>University of Reading

MIke, the DNA should be easier to dissolve at a slightly alkaline pH, (>pH8.0).  
If there is not enough buffering capacity, your solution may be below pH8.0, 
and this reduces the solubility of the DNA.  Try resuspending in a buffer
with a higher pH; after it is in solution (it may still take an overnight 
incubation), adjust the pH back to neutral.
Janet Braam
Dept. Biochemistry and Cell Biology
Rice University
Organization: Penn State University
Date: Tuesday, 14 Apr 1992 17:06:32 EDT
From: <CSB3@psuvm.psu.edu>
Newsgroups: bionet.molbio.methds-reagnts
Subject: In search of ergothioneine

I'm in desperate need of some ergothioneine. If anyone knows how or where to ge
t some, please let me know. Call Dennis (814)865-1247.
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: DIGESTION OF PCR PRODUCTS
From: falquet@sc2a.unige.ch
Date: 9 Apr 92 15:20:45 +0200
Distribution: bionet
Organization: University of Geneva, Switzerland

In article <9204010949.AA16192@genbank.bio.net>, UDHA094@OAK.CC.KCL.AC.UK (MARCEL KUIPER) writes:
> 
> Dear colleagues,
> 
> 
> I am trying to introduce restriction sites at the 5' and 3' end of PCR
> products, to ligate the digested product in a vector. Until now I've
> been unsuccesfull in doing so. At the 5' end I've introduced a KpnI site
> with 6 bp in front of it; at the 3' end I've introduced a BglII site with
> 2 bp extra at the 3' end (should be sufficient according to the New England
> Catalog).
> After PCR, I treat the products with Proteinase K to remove Taq polymerase
> sticking to the ends, and digest the product with the restriction enzymes
> and ligate it into a vector digested with KpnI and BglII.
> Unfortunately, the transformed ligation product doesn't yield any colonies.
> Can someone help me solve this problem?
> 
> Thanks very much in advance,
> 
> 
> Marcel Kuiper.

It is always difficult to cut close to the ends of a PCR product.

Try: 
- Kinase the ends (Oligos for PCR don't have 5' phosphates)
- Blunt end your PCR products with Klenow 
- Blunt ligate into a blunt cutted/cipped vector
- Transform, miniprep, digest with KpnI and BglII (this time they cut fine)
- Low melt purify your fragment 
- Ligate into the final vector between KpnI & BglII sites.

It is longer but it works...

Laurent FALQUET and Eduardo GONZALEZ (U.K. Laemmli group)


Newsgroups: bionet.molbio.methds-reagnts
From: kseiden@casbah.acns.nwu.edu (Kenneth Seidenman)
Subject: Re: Preparation of Genomic DNA
Organization: Northwestern University, Evanston, Illinois
Distribution: bionet
Date: Thu, 9 Apr 1992 16:31:33 GMT

Hi,

I was wondering if anyone had a good protocol for preparing genomic DNA from
frozen liver tissue.  Since this DNA will be used for PCR, I'm particularly
concerned about cross-contamination between samples during the "pulverization"
steps.  I tried using a hammer and some tin foil, but I found this to be
rather messy and think there's a fair chance of cross-contamination.  Any
suggestions would be appreciated.

Thanks

Ken
From: nigel@codon3.berkeley.edu
Newsgroups: bionet.molbio.methds-reagnts
Subject: YCp50: can someone point me to the sequence?
Keywords: YCp50, sequence
Date: 7 Apr 92 16:37:45 GMT
Article-I.D.: agate.rsj4pINNb1b
Reply-To: nigel@codon3.berkeley.edu ()
Distribution: bionet
Organization: University of California, Berkeley
NNTP-Posting-Host: codon3.berkeley.edu

Hi!

The header pretty much says it all.  I'm trying to find the sequence of
YCp50 (yeast centromeric plasmid), and had no luck with Embl or GenBank.
I'm perfectly capable of dredging up the original reference, and piecing
the sequence together from scratch.... I just thought I'd try here first.

thanx

-nigel

BTW: The person who gave this vector to me said it was so common that I could
     find a map for it in the NEB catalogue (I couldn't)... who does sell it?
From: C749SCB@SEMOVM.SEMO.EDU (Walt Lilly)
Newsgroups: bionet.molbio.methds-reagnts
Subject: protease mutants in fungi
Date: 7 Apr 92 22:28:55 GMT
Distribution: bionet

We are trying to isolate mutants of a wood-decaying fungus that are
deficient in specific proteolytic enzyme activities.  Can anyone
offer advice on a selection scheme (using liquid media or plates)
which might allow us to find such mutants?  We know that the proposition
will be complicated by the fact that the fungus produces and secretes
several different proteases, including both endo- and exo- peptidases.

Does anyone know of any commercially available peptides conjugated to
a toxin....this seems like it might be an approach to select only for
protease- mutants.  Any thoughts?

************************************************************************
*  WALT LILLY                     *                                    *
*  DEPARTMENT OF BIOLOGY          *  INTERNET: C749SCB@SEMOVM.SEMO.EDU *
*  SOUTHEAST MISSOURI STATE UNIV. *  BITNET: C749SCB@SEMOVM            *
*  CAPE GIRARDEAU, MO 63701       *                                    *
************************************************************************
From: BOROS@OHSTMVSA.BITNET
Newsgroups: bionet.molbio.methds-reagnts
Subject: A quick help is needed from Ohio with Western blotting!
Date: 15 Apr 92 19:50:00 GMT
Distribution: bionet


We are running 2 SDS-PAGE gels and need to blot both but we only
have one transblot chamber. Will soaking one gel over night affect
the proteins (i.e. will they diffuse out much?).
Any help is very much appreciated. This thing is hurry becouse we
have one hour left till the end of running the gels and we
have to figure out what to do whit one of the gels. It is 3:45 PM
in Ohio. Thanks, Laszlo
..
                                                              ...
       *************************(O)******************************
       * Laszlo G. Boros, MD                                    *
       * Research Associate - 2B/H                              *
       *                                                        *
       * The Ohio State University College of Medicine          *
       * Department of Surgery                                  *
       * 400 West 12th Ave. Wiseman Hall Room No.217            *
       * Columbus, Ohio 43210                                   *
       * U.S.A.                                                 *
       *                                                        *
       * Telephone: (614) 292 7066 - business                   *
       *        &   (614) 459 2942 - home                       *
       *       Fax: (614) 293 4063 - business                   *
       *                                                        *
       * E-mail:    BOROS@OHSTMVSA.ACS.OHIO-STATE.EDU           *
       *            BOROSL@MAGNUS.ACS.OHIO-STATE.EDU            *
       *                                                        *
       *************************(O)******************************