HDTenEightyP/NetNews huggingface dataset:
From: a52041@sakura.kudpc.kyoto-u.ac.jp (Michael CHENG)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Baculovirus
Date: 7 Nov 92 00:58:16 GMT
Organization: Data Processing Center, Kyoto University, Kyoto Japan.
Originator: a52041@sakura.kudpc.kyoto-u.ac.jp

From: a52041@sakura.kudpc.kyoto-u.ac.jp (Michael CHENG)
Newsgroups: bionet.molbio.methds.reagnts
Subject: Baculovirus
Summary: 
Followup-To: 
Distribution: world
Organization: Data Processing Center, Kyoto University, Kyoto Japan.
Keywords: Baculovirus

Hi! I am wondering if anyone out there has experience using the BaculoGold
Transfection Kit manufactured by Pharmingen. Our lab is planning on purcahsing
it soon but would like to get as much comment as we can due to its high price.
  We have previously tried using the vector pAcYM1 but have not yet been able
to isolate any recombinant virus yet. Can someone also give us some reference
as to how to perform a PCR to confirm that a homologous recombination effect
has taken place?

Many Thanks!


Michael CHENG
Dept. of Biological Chemistry
Faculty of Pharmaceutical Sciences
Kyoto University

a52041@sakura.kudpc.kyoto-u.ac.jp
From: pnh@fcsparc6.ncifcrf.gov (Paul N Hengen)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: FAQ's and ?moderator
Summary: FAQ ftp site
Date: 6 Nov 92 18:04:54 GMT
Distribution: bionet
Organization: Frederick Cancer Research and Development Center

btroen@uv1.im.med.umich.edu writes:

> Is there a server for the FAQ's?  And what topics do they cover?

Our news reader/poster has been under reconstruction and some
of my postings were not getting through. I tried to post the
FAQ list twice, but am still not sure if it went out from here.
If the posting has not gone out, someone please tell me and I'll
post it again.
 
The FAQ list is available by anonymous ftp to ncifcrf.gov
Simply get the file pub/methods/FAQlist

I plan to update the FAQ list whenever possible and place the newest 
version in the ftp site here. Do other people on the net want me to
post this FAQ list on bionet.molbio.methds-reagnts regularly?

The current questions within the FAQ list are:

================================================================================
 1. What are some good reference books for methods in molecular biology?
 2. Where can I get information about the genotypes and phenotypes of
    common E. coli strains used in molecular genetics?
 3. How can I access MEDLINE?
 4. How do I amplify pBR322 plasmid using chloramphenicol?
 5. If a plasmid has more than one site for a particular restriction
    enzyme, is there some way to get the enzyme to cut at only one site?
 6. How do I calibrate a Pipetman(tm) style micropipette?
 7. Is it possible to get a personal copy of the files stored at Genbank?
 8. How I do prepare powdered silica for DNA purification, with the
    associated solutions?
 9. How do I use powdered silica to isolate DNA from agarose gels?
10. How do I use silica powder to prepare plasmid DNA for sequencing?
11. What is PCR?
12. What are some good reference books for PCR?
13. How should I select a set of primers to use for PCR?
14. What kinds of programs are available for designing PCR primers?
15. What is "Hot-start" PCR?
16. What is AP-PCR or RAPD PCR?
17. What is "Touchdown" PCR?
================================================================================

If anyone would like to make additions or corrections to
the FAQ list, please send the information to me at pnh@ncifcrf.gov

Paul N. Hengen
National Cancer Institute
Frederick Cancer Research and Development Center
Frederick, Maryland 21702-1201 USA
From: adavies@vax.oxford.ac.uk (Anthony Davies)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Baculovirus
Date: 9 Nov 92 21:20:20 GMT
Organization: Institute of Virology, Oxford

In article <1992Nov7.005816.9346@kudpc.kyoto-u.ac.jp>, a52041@sakura.kudpc.kyoto-u.ac.jp (Michael CHENG) writes:
> Hi! I am wondering if anyone out there has experience using the BaculoGold
> Transfection Kit manufactured by Pharmingen. Our lab is planning on purcahsing
> it soon but would like to get as much comment as we can due to its high price.
>   We have previously tried using the vector pAcYM1 but have not yet been able
> to isolate any recombinant virus yet. Can someone also give us some reference
> as to how to perform a PCR to confirm that a homologous recombination effect
> has taken place?

My strong advice is not to go with Pharmingen's product, but with a kit 
produced by Clontech (called BakPak :-).  The double-cut baculovirus DNA 
marketed by Pharmingen was developed in, and licensed from, this Institute.  
It is a magnificent system - anyone can now *routinely* make recombinant 
baculoviruses....it is easier than cloning!  However, Pharmingen only sell 
the pure, double-cut baculo DNA - essentially a disposable item.  Clontech, 
however, sell the intact baculovirus as well, from cultures of which you can 
purify and double cut as much DNA as you ever want.  Got to be a better deal!  

Actually Pharmingen put pressure on Clontech NOT to do this, but to their 
credit they held out and will continue to produce it.  Also, Paul Kitts, 
who developed the system while here, now works at Clontech, so I can 
promise you'll get 1st rate technical support from him.

On the subject of PCR, Clontech also supply oligos for the purpose you have in 
mind as part of the BakPak.

Happy Trekking!!
________________________________________________________________________________

Anthony Davies                Voice: 865-512361
Institute of Virology         Fax:   865-59962
Mansfield Road                Vax:   adavies@vax.path.ox.uk.ac (preferred)
Oxford OX1 3SR                       adavies@vax.ox.uk.ac (this account)
UK
________________________________________________________________________________
From: ram3@po.CWRU.Edu (Russell A. Maurer)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: PCR Mutagenesis
Date: 13 Nov 92 05:10:35 GMT
Reply-To: ram3@po.CWRU.Edu (Russell A. Maurer)
Organization: Case Western Reserve University, Cleveland, OH (USA)
NNTP-Posting-Host: slc12.ins.cwru.edu


In a previous article, bhjelle@carina.unm.edu () says:

>I encountered an impressively high level of mutagenesis (14 
>errors out of 411bp) in amplifying a segment of DNA from
>a plasmid clone. All mutations involved T or A in some
>manner. 11 were point mutations (transitions and
>transversions), but 3 were 1 bp insertions or deletions.
>
>This mutation rate is much higher than anything I or
>anyone else I talked to had encountered with Taq polymerase.
>A colleague suggests that the problem may have been due to
>the rapid loss of nucleotides in the first few cycles of PCR
>related to using an exceptionally large amount of template
>plasmid DNA (I used about 50ng). He thinks that when Taq
>tries to copy the DNA but has run out of dNTPs, it might
>put in the wrong base. It has no proofreading function.
>
>This sounds plausible to me, but unfortunately I just
>generated a bunch of new TA clones using a large amount of
>plasmid templates and am reluctant to go back and do that
>again on the assumption that they are badly mutated too.
>
>If this is indeed the explanation, it seems to me that
>some clever soul might have exploited it to develop a PCR-based
>random mutagenesis strategy. Do any of you hard-core
>NAR readers know about anything like that? 
>
>Any other ideas appreciated.
>
>Brian
>
>
See Leung, D., E. Chen and D. Goeddal, Technique 1:11-15 (1989) for random
mutagenesis by PCR in the presence of manganese and Y. Zhou, X. Zhang and R.H.
Ebright, Nucleic Acids Research 19:6052 (1991) for random mutagenesis under
essentially standard (magnesium) PCR conditions.  Ebright reports that about
35% of PCR clones sequenced at random carried a mutation over a 633-base
interval.  I have not tried it myself.

Russ Maurer>
>
>
>
Newsgroups: bionet.molbio.methds-reagnts
From: cbeall@magnus.acs.ohio-state.edu (Clifford J Beall)
Subject: Re: Baculovirus
Organization: The Ohio State University
Date: Tue, 10 Nov 1992 19:08:23 GMT

                              Cliff Beall
                              Ohio State Biotech Center
Newsgroups: bionet.molbio.methds-reagnts
From: S.Yunchalard@massey.ac.nz (S. Yunchalard)
Subject: Re: lactococcus media
Summary: lactococcus lactis culture
Keywords: media
Organization: Massey University, Palmerston North, New Zealand
X-Reader: NETNEWS/PC Version 2c
Date: Tue, 10 Nov 92 00:22:09 GMT

For routinely cultivation of Lactococcus lactis, M17 media 
(Terzaghi et al., 1975) can be used. This media is now 
commercial available from Gibco.

Best Wishes
Sirinda
From: MCAO@VTHVAX.TAMU.EDU
Newsgroups: bionet.molbio.methds-reagnts
Subject: Cause of phage difference?
Date: 10 Nov 92 16:25:36 GMT
Distribution: bionet


	Hi, the size of the phage on my screening plate varies a lot. What may
cause this phenomenon? The insert size or the bacterial strain?
						Ming Cao
Newsgroups: bionet.molbio.methds-reagnts
From: jgraham@bronze.ucs.indiana.edu (the End)
Subject: Sequences of pMC9, pMC1403, or pSKS105 ?
Organization: Indiana University
Date: Thu, 12 Nov 1992 22:52:46 GMT

I have a graphic map of pSKS105 with many sites which leads me to think
that the entire sequence is known, yet GenBank searches give me no matches,
including the parent plasmid pMC1403. I cannot find any of the pMC plasmid
sequences either (pMC9, pMC1, ect.) Any pointers would be much appreciated.

Thanks,

Jim
J. Graham
From: etaylor@ccu.umanitoba.ca (Euan R. Taylor)
Newsgroups: bionet.molbio.methds-reagnts
Subject: immunopptn of RNA/ribosome complexes
Date: 12 Nov 92 21:43:48 GMT
Organization: University of Manitoba, Winnipeg, Canada

Dear all,
Has anyone out there had experience with isolation of RNA's for specific
products using immunopreipitation of RNA/ribosome complexes?
I know this has been done, but I dont remember (of course) any specific papers.
I am trying to isolate a product by antibody screening of a gt22 library,
however luk has not so far been with me and this is a contingency plan..
Any advice or references would be welcome
ta
Euan Taylor
etaylor@ccu.umanitoba.ca
From: wetsel_r@wums.wustl.edu
Newsgroups: bionet.molbio.methds-reagnts
Subject: Gel Slick anyone?
Date: 13 NOV 92 00:07:47 GMT
Organization: MIT PLASMA FUSION CENTER
NNTP-Posting-Host: msnews.wustl.edu

Greetings:

My wife and I (work in the same lab :-) ) were curious if anyone has
tried the AT Biochem Gel Slick as an alternative to Pierce's Surfacil.
Pierce's product works well but the HCl fumes generated can be irratating.

David
haviland@kids.wustl.edu
From: btroen@uv1.im.med.umich.edu
Newsgroups: bionet.molbio.methds-reagnts
Subject: FAQ's and ?moderator
Date: 6 Nov 1992 15:48:12 GMT
Organization: University of Michigan
Distribution: usa
NNTP-Posting-Host: btroen.im.med.umich.edu

  Sorry for these simple questions, but I just started subscribing:

	1)  Is there a server for the FAQ's?  And what topics do they cover?

	2)  Who is the moderator of this group and whence does it originate?
From: cytogen@chmeds.ac.nz (Martin Kennedy)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: stable transfection
Date: 12 Nov 92 05:10:32 GMT

In article <40729@sdcc12.ucsd.edu>, wsun@jeeves.ucsd.edu (Fiberman) writes:
> Hello Netter,
> 
> I am going to use the pRc/CMV vector from Invitrogen to express a cDNA
> in HELA cells.  I want to select for the transfected cells with
> neomycin resistance and establish a stably transfected cell line
> expressing my clone.  Can anyone out there recommend a good reference,
> or give some helpful tips on how to proceed?  Any suggestions
> would be appreciated.
> 
> -fm

First of all, determine the G418 sensitivity of Hela cells with your batch of
G418 and in your culture system.  Culturing in medium containing from 100ug/ml
up to 1mg/ml of G418 should reveal the best lowest conc. that causes killing. 
G418 takes a few days to kill cells.

Then make a large (preferably cesium, but other prep methods work) plasmid
prep, and linearize your construct anywhere in the vector sequences.  Then
transfect by whatever method works best for Hela cells, and place cells under
selection about 24 or 48 hours later.  You should get resistant colonies in
about a week or two.  You can pick these manually in several ways.  I use a
flame drawn, bent pasteur pipette, with mouth suction device (and cotton wool
filter).  Replace the medium in your plates with PBS.  Suck up some trypsin in
the pipette, and (with a binoc microscope) carefully eject the trypsin over and
around the colony to be picked. Scratch of the colony, and suck up the cells. 
Transfer to trypsin, until single cells obtained, and then plate back into
selective medium on 24-well plates.> 

Some useful references are:

Michael Kriegler:  Gene transfer and expression; a laboratory manual. Stockton
Press, 1990.

E.J.Murray (ed):  Gene transfer and expression protocols. Methods in Molecular
Biology Vol 7.  Humana Press 1991.

	(Especially Chapter 19, R.F.Santerre et al., Use of vectors to confer
resistance to antibiotics G418 and hygromycin in stably transfected cell lines;
pp245-256).

Hope this is useful,

Cheers,
Martin
Newsgroups: bionet.molbio.methds-reagnts
From: morrisj@wsuaix.csc.wsu.edu (John Morris;S23115)
Subject: Re: sequencing from 300-500 bp
Organization: Washington State University
Date: Fri, 13 Nov 92 03:41:58 GMT

In article <1992Nov12.150858.16911@magnus.acs.ohio-state.edu> rrumpf@magnus.acs.ohio-state.edu (Robert Rumpf) writes:
>There is a product called "Long Ranger" which substitutes for the acrylamide 
>gels.  It is supposed to do exactly what you asked for, give resolution on the 
>order of 500-800 (?? been a while since I've heard about it) bases per gel.  
>Several people in our department have tried it; only one has gotten it to work.
>Everyone else had technical problems either pouring such a gel or taking it off
>the glass.
>
>Bob

I've had good success with "Long Ranger", but one does need to cool the plates
to room temp before pulling them apart.

John Morris, IBC, Washington State Univ.
From: preissj@CLVAX1.CL.MSU.EDU ("J Preiss--Seq Anal")
Newsgroups: bionet.molbio.methds-reagnts
Subject: re in vitro mutagenesis kits
Date: 12 Nov 92 13:37:00 GMT
Distribution: bionet

In response to the recent request for recomendations for a favorite in
vitro mutagenasis kit, our favorite here is the Amersham kit for site
directed mutagenasis.  
	We have used the old Kunkel method and the s
Stratagene method with less success.  The Amersham kit seems very easy and
so efficient that we have started bulking several primers per reaction to
save on reagents, and we still get all our mutants.

		Good Luck

Dr. Leonard N. Bloksberg
PreissJ@clvax1.cl.msu.edu

Dept. of Biochemistry
Michigan State University


Newsgroups: bionet.molbio.methds-reagnts
From: jgraham@bronze.ucs.indiana.edu (the End)
Subject: Re: PCR Mutagenesis
Organization: Indiana University
Date: Fri, 13 Nov 1992 15:06:31 GMT

In <mxdq3j#@lynx.unm.edu> bhjelle@carina.unm.edu () writes:

>I encountered an impressively high level of mutagenesis (14 
>errors out of 411bp) in amplifying a segment of DNA from
>a plasmid clone. All mutations involved T or A in some
>manner. 11 were point mutations (transitions and
>transversions), but 3 were 1 bp insertions or deletions.

>Brian

Very interesting and disturbing. We routinely generate templates for in vitro
transcription via PCR using a protocol (Higuchi et al. NAR 16 (15) p.7351) 
recomended by a well-known colleague.

This procedure uses plasmid targets at 200-400 ng/ rxn. with dNTPs at 0.175 mM
and a mere 16 cycles. The low cycle number is suggested in order to 
reduce the chance of errors generated by Taq. I obtain about 1 to 5 ug of
500 bp fragments from target plasmids around 3 Kb.

In the NAR paper describing this technique, Krummel and associates report that
they have not seen a single base change in preparing 10 different transcription
templates in the 500 bp size range (p. 7359 line 14).

Are you using a significantly lower dNTP concentration ?


Jim
J. Graham
Biology/Chemistry Depts.
Indiana Univeristy -Bloomington
......................."Just saying NO to kits"................................
From: wetsel_r@wums.wustl.edu
Newsgroups: bionet.molbio.methds-reagnts
Subject: Oligos for sequencing...
Date: 11 Nov 92 12:16:30 GMT
Organization: MIT PLASMA FUSION CENTER
NNTP-Posting-Host: msnews.wustl.edu

Hi Netters:

This may be a common theme or even an FAQ, but since I don't know I'll
ask anyway.  To those that make their own oligos and do double stranded
sequencing, what protocols do  you use, if any, in processing your oligo
after it has been deprotected?  EtOH PPTs? Phenol?

David
haviland@kids.wustl.edu
From: suter@VAX.MPIZ-KOELN.MPG.dbp.de
Newsgroups: bionet.molbio.methds-reagnts
Subject: re: hyb problems
Date: 11 Nov 92 11:50:11 GMT
Distribution: bionet
Content-Identifier: 12
Original-To: methods@uk.ac.daresbury
Conversion: Prohibited

+> Help!  I am seeing a very intense background in all my blots, recently.  The
+
+.....stuff deleted...
+>
+> I changed blot (to gene screen plus), changed probe and even used a different
+> pre hyb solution (5x SSC, 5x denhart, 0.5%SDS, 20mM phosphate buffer, 300u/ml
+> salmon sperm DNA, 5mM EDTA), but nothing seems to eliminate the very intense
+> background.  I have even tried washing with water.

+I find GeneScreen Plus requires higher concentrations of salmon sperm DNA for
+blocking than several other membranes.  If the black background doesn't wash
+off then it's covalently bound to your membrane, and the membrane probably
+wasn't sufficiently blocked in the first place.  Try using ca 500ug/ml ssDNA in
+your prehyb mix; I also add a further 1-2mg of ssDNA to my probe when I boil
+it, and then add about 10ml of hyb mix before tipping it into the bag/bottle.
+As for the black membranes, put them away for a few months!
+
+> Jaishree [jaishre@matt.ksu.ksu.edu or jaishre@ksuvm.ksu.edu]
+>
+Good luck!
+
+Martin

a very effecient blocking agent is SDS, we use it at conc of 7 % in our hyb
mix (this is essential for zetaprobe by the way). for washing we use
1 x SSPE, 1% SDS. These rather high concentrations have very relevant effects !
 
cheers,

clemens
===============================================================================
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. xx49-221-5062.21
suter@vax.mpiz-koeln.mpg.dbp.de
===============================================================================
       all job offers should include a self-addressed, stamped envelope
===============================================================================
From: jgraham@bronze.ucs.indiana.edu (the End)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Nested deletion kits/methods
Date: 8 Nov 92 17:52:41 GMT
Distribution: bionet
Organization: Indiana University


Forget the stupid kit. Just make three buffers as described in Maniatis,
get ExoIII and Mung Bean Nuclease. This affection for pre-packaged "kits"
is ridiculous and is actually harmful,increasing research costs and hurting
the exchange of information between reseachers.

By the way, my exo III deletions were always clustered at specific sites,
often at the same residue. In the 20 or so products I sequenced, I only found 
a single BIdirectional deletion of an inappropriate size. For producing a 
set of bidirectional deletions in the range of 10 to 60 nt I found the 
Exo III procedure would not generate a continuous range of centered deletions
in my target sequence. I increased nuclease concentrations up to 30-fold above 
those recomended, and still observed only unidirectional deletions after 
generating a pair of suitable ends.

If anyone has had luck making bidirectional deletions, please contact me.


Thanks,

Jim
J. Graham
Biology/Chemistry Dept.
Indiana University -Bloomington
********************************* "Just say NO to kits !" *******************
From: mic268a@vaxc.cc.monash.edu.au
Newsgroups: bionet.molbio.methds-reagnts
Subject: lactococcus media
Date: 5 Nov 92 23:31:34 GMT
Organization: Computer Centre, Monash University, Australia


Hi there,

Is there anyone out there who routinely cultures Lactococcus lactis?
If so I was wondering if you could supply me with a recipe for the media used
to grow this bug.

Thanks in advance
Mike L.
Monash University
AUSTRALIA
From: axa12@po.cwru.edu (Ashok Aiyar)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: MEDLINE
Date: Sat, 7 Nov 1992 22:19:32 GMT
Organization: CWRU School of Medicine
Distribution: bionet
NNTP-Posting-Host: axa12-slip.dialin.cwru.edu

In article <49462.leonfdo@ccwf.cc.utexas.edu> leonfdo@CCWF.CC.UTEXAS.EDU (@ccwf.cc.utexas.edu, Leon F. Garcia) writes:

>This may be common knowledge, or it may have been asked before, but I don't\
>know how to access, if possible, MEDLINE via gopher or telnet (preferably for
>free). I would if somebody out there knows how to do this please let me know

MEDLINE can be accessed over the Internet (via Telnet) but I do not believe 
that access is free (at least we still pay for it).  For those who are 
interested, the NLM MedLine "search" software works fine over SLIP 
connections.  I just got it working about 10 minutes back.  The trick is to 
use the ETHERSLIP packet driver and not the SLIP8250 driver (on DOS 
machines).

Ashok
----------------------------------------------------------------------
                             Ashok Aiyar
                     Department of Biochemistry
                         CWRU Medical School
                          axa12@po.cwru.edu
----------------------------------------------------------------------
From: MPB@CBR.DWE.CSIRO.AU (Mark Bradley)
Newsgroups: bionet.molbio.methds-reagnts
Subject: RE: Dewaxing
Date: 11 Nov 92 11:42:53 GMT
Distribution: bionet

why not try the routine "old fashion" method of dewaxing with Xylol?
Any standard histo text will outline the method.
From: jjc4@po.CWRU.Edu (James J. Campanella)
Newsgroups: bionet.molbio.methds-reagnts
Subject: nonlethal gus assay
Date: 13 Nov 92 22:26:09 GMT
Organization: Case Western Reserve University, Cleveland, Ohio (USA)
NNTP-Posting-Host: thor.ins.cwru.edu


Question to the net:

I have been trying to co-transform an arabidopsis cell line with

a gene I'm interested in and a gus marker. I can assay for the

gus, but then I may lose a transformant because I sacrifice the

colony to the substrate assay. My question is: is there a non-lethal

way to assay gus in plant cells, so that they can survive to be

placed back on agar and grown further? thanks.
-- 
---------------------------------------------------------------
Jim Campanella   | "If one strives for mediocrity, then the
Case Western     |  worst thing that may happen is that one will
University       |  succeed."  -Buckminster Fuller
From: jgraham@bronze.ucs.indiana.edu (the End)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: PCR Mutagenesis
Date: 13 Nov 92 18:04:45 GMT
Organization: Indiana University


I couldn't find but one other reference to the use of PCR in random mutagensis,
Journal of Biological Chemistry 265 (33)p20085. It just shows that 25 cycles
on a 480bp fragment generated phenotypic mutants at around 4%. This of course
does not indicated changes at the DNA level.


If anyone has access to the now famous journal "Technique", could you please
post the Mn+ levels used to decrease Taq fidelity ? If possible the reaction
conditions used and the resulting misincorporation statistics ?

Thanks much,

Jim
J. Graham
Biology/Chemistry Departments
Indiana University -Bloomington