HDTenEightyP/NetNews huggingface dataset:
From: djt2@po.CWRU.Edu (Dennis J. Templeton)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Mac Word and Excel templates for NIH grant forms-(about)
Date: 3 Sep 1992 22:33:19 GMT
Organization: Case Western Reserve University, Cleveland, OH (USA)
Reply-To: djt2@po.CWRU.Edu (Dennis J. Templeton)
NNTP-Posting-Host: thor.ins.cwru.edu


Recently someone posted a request for NIH grant form (PHS398) in electronic
format.  

In the following posting is a copy of the version I posted a few months ago. 
It is a 39k file in binhex/stuffit form that yields the templates for the
new/competing form in Word 5 version and budget templates in Excel 2.2

The Word 5 templates translate well to Word For Windshields and I assume
that the Excel files could be updated to Excel 4 too... since my own grant
came in recently I can *maybe* afford an upgrade.

Good luck, and disseminate these as far as you like.  Keep the parts and
instructions together, though, since the  Excel files are linked.

P.S. make a COPY before you modify them, or at least don't ask me to send
replacements!

The file is in the following post.

Dennis Templeton
From: bhjelle@carina.unm.edu
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: DNA labelling kits
Date: 4 Sep 92 20:30:56 GMT
Organization: University of New Mexico, Albuquerque

In article <1992Sep4.115359.1633@gnv.ifas.ufl.edu> afc@gnv.ifas.ufl.edu writes:
>A few years ago we switched from labelling by nick translation to
>labelling by random priming.  We use BRL kits for both.  Besides 
>being easier to use and more reliable, random priming gives about
>100x higher specific activity.   Mostly we are probing total cell
>DNA (from single mosquitoes) for mitochondrial or ribosomal genes, 
>so probe should be in excess.
>
>My question is: why don't we see a 100x increase in hybridization
>intensity?  RP probes give a little better results than NT probes,
>maybe 2x or 3x in our hands.  
>
>I am guessing that probe length might be a problem: NT probes have a
>several hundred bases of unlabelled DNA with the labelled DNA
>attached.  RP probes have six unlabelled bases attached to the label.
>Depending on how long the synthesized strands are, this could make a
>big difference.  If so, would playing with primer concentration help?
>What about other labelling kits?
>
I had a lot of problems with random hexamer priming. Exactly
as you describe. You get unbelievable specific activity.
In my case, probing mammalian DNA, some genes would produce
no bands or only very faint bands despite the great specific
activity. In my case, this result was much worse than I
had obtained with the more humble nick-translated probes.

I always assumed it was due to the multiple priming events that
were a very short distance apart. When I ran the probe on
a gel, it was composed of very small fragments. This might
be fixable by lowering the stringency of hybridization and
washing,  or by priming with, say, 10mers, but I just moved
on to RNA probes which worked better in my hands than any
DNA probe.

If someone tells you that they are getting good results with
a particular labeling protocol, always do the following:

1. Ask them what kind of organism they are probing. What works
for phage, E. coli, or yeast may not work well for insects.

2. If the genome size of their organism is >= yours, get their
recipe.

Brian
Newsgroups: bionet.molbio.methds-reagnts
From: cdds01@vaxa.strath.ac.uk
Subject: Strange receptor binding studies, or are they?
Organization: Strathclyde University VAX Cluster
Date: Wed, 26 Aug 1992 13:42:34 GMT

I have recently been doing some receptor-binding studies (growth hormone binding to
lymphocytes). I add my 125Iodinated tracer and add cold competitor to my cells +
tracer. After a suitable incubation period I harvest the cells by centrifugation
then count the pellet. The thing is the counts I get back show that tracer binding
increases as I add cold competitor (instead of decreasing as I expected). Does anyone
have any ideas on why this should happen?

Thanks in advance
Pete Crilly
From: ser3@CUNIXF.CC.COLUMBIA.EDU (Stuart E Ravnik)
Newsgroups: bionet.molbio.methds-reagnts
Subject: genbank e-mail searching
Date: 25 Aug 92 18:41:36 GMT
Distribution: bionet



please send me info on searching genbank  for homology/alignments via
e-mail.

Also, any info on the NCBI server recently initiated to replace
genbank.


Muchos gracias

Stuart E. Ravnik
ser3@cunixf.columbia.cc.edu
Newsgroups: bionet.molbio.methds-reagnts
From: wakarchu@biologysx.lan.nrc.ca (Dr. Warren Wakarchuk)
Subject: Re: hydrolysis of hydrophobic proteins
Organization: National Research Council of Canada
Date: Thu, 3 Sep 92 21:05:08 GMT

In article <185492INNq2m@matt.ksu.ksu.edu> drintoul@matt.ksu.ksu.edu (David A Rintoul) writes:
>From: drintoul@matt.ksu.ksu.edu (David A Rintoul)
>Subject: hydrolysis of hydrophobic proteins
>Date: 3 Sep 92 13:34:26 GMT

>Greetings,

>  this may not be the classis topic for this question, but I
>am dealing with a biological molecule, at any rate.

>We have purified a protein from lens fiber cell plasma membranes 
stuff deleted...

>So, the real questions are:

>1. Can we do the hydrolysis on protein that has not been dried down,
>but still contains a bit of chloroform and methanol?  

>2. Does anyone out there have experience with such hydrophobic
>proteins?  Any hints?  

>This analysis is the last thing we need to do before writing up the
>paper, and it is getting frustrating that we seem unable to get it
>done.  Any clues or suggestions will be greatly appreciated.

>P.S.  We have considered SDS-PAGE and blotting to PVDF, followed
>by AA analysis, but, since the purpose of this whole escapade
>is to quantitate the protein from the AA content, I don't think 
>this is a good (quantitative) approach.

>Dave Rintoul            Internet: drintoul@matt.ksu.ksu.edu

I have no experience with such hydrophobic proteins, but I can make a few 
suggestions for your analysis.  In the newer amino acid analyzers (ABI) the 
hydrolysis is performed at higher temperatures 150 C for short times, which 
may help you.  The higher temperature/short times have also been used 
for manual hydrolysis, but you will loose Ser/Thr/Tyr. Traces of CHCl3 and 
methanol are likely not going to interfere with the hydrolysis, but you 
might esterify some carboxyl groups.  Actually people do the hyrolysis for 
as long as 72 h at 110 C, to cleave the hydrophobic linkages. 
 Your idea of PVDF blots is actually not bad, but there are some artifacts 
generated here from contaminants.  Can you calculate an extinction 
coefficent and quantify the protein that way?

Warren Wakarchuk IBS NRC  wakarchu@biologysx.lan.nrc.ca
From: bhjelle@carina.unm.edu
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: PBR322 - maximizing yields
Date: 28 Aug 92 19:30:56 GMT
Distribution: bionet
Organization: University of New Mexico, Albuquerque

In article <3778@fcs280s.ncifcrf.gov> pnh@fcs260c2.ncifcrf.gov (Paul N Hengen) writes:
>>bhjelle@carina.unm.edu () writes:
>
>:Unfortunately, some things can only be done in pBR-based
>:vectors and I have been plagued with totally miserable
>:yields of one particular example of same for more than
>:a month now.
>
>I have two thoughts about your problem...
>
>1. You may have cloned a segment of DNA that would prohibit
>high copy number in any plasmid, in which case you will be
>wasting much effort trying to amplify.
>
>2. Why not spend some time subcloning into one of the
>runaway plasmids that can be amplified simply by changing
>temperature? This could save quite a bit of aggravation
>and the bottle of chloramphenicol. See these refs...
>
>B. E. Uhlin et al. Gene 6:91-106. (1979)
>B. E. Uhlin et al. Gene 22:255-265. (1983)
>
Actually the plasmid is a trpE expression construct, and I
am having the most problems with the vector itself, rather than
derivitives containing my inserts. The lab I got the clone from
swear that it grows about as well as pBR, and that there is
enough tryptophan in LB to repress the trp E anyway.

I could move the whole trpE gene into pUC I suppose, but that
would require being able to grow the pBR-based vector.

Brian
From: ludwig@biology.ucsc.edu (Bob Ludwig)
Newsgroups: bionet.molbio.methds-reagnts
Subject: VisiGel
Date: 3 Sep 92 18:24:09 GMT
Distribution: bionet


Does anyone know the macromolecular nature of VisiGel, Stratagene's 
matrix for resolving low MW DNA fragments?

Thanks.

Bob Ludwig
Sinsheimer Labs
university of California
Santa Cruz

rludwig@cats.ucsc.edu
From: BISHOP@MSVAX.MSSM.EDU ("DAVID F. BISHOP")
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Tm
Date: 28 Aug 92 22:23:50 GMT
Distribution: bionet


Andrew Cockburn asks:

>>Wetmur, James G. DNA probes:Applications of the principles of mucleic acid
>>hybridization. Crit. Rev. in Biochem. Mol. Biol. 26:277-259,1991.
>                                                     ^^^^^^^
>Is this an antisense article?

>Seriously, could you repost the (corrected) reference?  I for one would like
>to look at it.
>
>Thanks,
>Andrew Cockburn

Ah yes, those PCR (pretty cockeyed reading) errors again.  The correct
reference is:

    Crit. Rev. in Biochem. Mol. Biol. 26:227-259,1991
                                          ^ 

-David Bishop
From: BMA14432@VAX1.UTULSA.EDU
Newsgroups: bionet.molbio.methds-reagnts
Subject: Summary-PCR few cells
Date: 31 Aug 92 14:29:38 GMT
Distribution: bionet

Well, it looks like all the votes are in so here it is.
It seems most of the protocols are pretty similar.
They all follow the basic spin down cells, wash in buffer of choice(PBS,TBS ect)then lyse cells. How they go about this is somewhat varied. One suggestion is to dry cells then all a 5x mix(1 MTris-Cl 8.5
250 ul 1MKCl 188 ul, 1 M MgCl2 15 ul, 1 M DTT 50 ul, 2.9 ug/ul BSA 125 ul
NP-40(your detergent) 125 ul, H2O to 1000 ul total. Incubate 37 C 10 min.
 then add 5 ul of 10x PCR Buffer, 1 ul each primer(O.D.=0.5)
2 ul 10 mM dNTPs, 0.5 ul Taq(2.5u)
35.5 ul H20. then cycle.  This does not call for any Proteinase K which 
makes the protocol more attractive in my opinion. Why? Well, I have
found that  even after inactivation the proteinase K still inhibits the 
PCR process(granted not to a large degree but if your going after a 
single copy gene form just a few cells it becomes a big deal.)
Another protocol calls for Proteinase K and SDS as the detergent. 
Here again I prefer not to use SDS because it seems to have an negative
effect on the PCR.  There is a protocol in PCR Protocols-A Guide to 
Methods and Applications. edited by Innis, Gelfand, Sninsky, and White
which I have used successfully however it seems to come up short
in my hands when the cell number drops lower (below 100 cells).

Hope this is enough of a summary, but if not feels free to contact me
via e-mail.

Mark Brudnak
University of Tulsa
INTERNET: BMA14432@VAX1.UTULSA.EDU
From: bsh@MED.PITT.EDU (Basavaraju Shankarappa)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: PBR322 - maximizing yields
Date: 27 Aug 92 15:16:41 GMT
Distribution: bionet

> The postings about how to get great yields of pUC are all fine
> and good, but in most cases we get enough DNA from a pUC
> miniprep to keep us happy for a long time. 
> 
> Unfortunately, some things can only be done in pBR-based
> vectors and I have been plagued with totally miserable
> yields of one particular example of same for more than
> a month now.
> 
> What are the best tricks for increasing yields of pBR-based
> vectors?
> 
> Brian
> 
I have had to face similar circumstances where I had to use the 
pBR322 based plasmids.  I found the following protocols to work
very well with the HIV genomic clone DNAs.  

I pick my colonies first thing in  the morning and incubate at 32C
until evening when I am ready to leave (about 8-10 hrs incubation).
I take out about 0.8 ml out of about 3 ml and add glycerol (15%) to 
store the cells and to the rest I have added anywhere from 25 to 150
ug per ml of chloramphenicol and let it grow overnight.  
By the next morning the cells are usually ready and I have had reasonably
good yields.  
Good luck.
Raj   Kit (!) Shankarappa.
Univ of Pittsburgh.
From: Klaus.Matthaei@anu.edu.au
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: N2 storage of murine cell-lines
Date: 26 Aug 92 22:28:33 GMT
Distribution: bionet

>I've had problems in successfully freezing macrophage cell cultures so they 
>can be successfully resuscitated. The problem does seem to lie in the actual freezing. The cells are harvested in the log phase, suspended in a glycerol freezing mixture at five-fold concentration then left overnight (well insulated) in a -70C freeze
>Can anybody suggest alternative procedures? The alternatives facing me at present are constructing a "freezing box" which will cool at the correct rate in a -70C freezer (feasible) or persuading the department to buy a commercial cell freezer (less f
>
>Sean Shaw
>Microbiology, Reading University
>skrshaw@sksscsc1.rdg.ac.uk


G'Day

Forget the need for commercial freezing machines.

For many years I have been able to successfully freeze a wide range of
mammalian cells including mouse and cattle cells, monoclonals etc. with
excellent retrieval rates (sometimes > than 90%). The cells are simply
suspended in sterile 90% Foetal Calf serum, 10% DMSO, frozen overnight at
-70*C in a styrofoam container and then stored in liquid nitrogen (some
have been down more than 10 years and still revive).  Retrieval is by quick
thawing in a 37*C water bath until JUST thawed (do not warm to 37) and
IMMEDIATE resuspension in fresh COLD medium (DO NOT pellet the cells before
resuspension in the fresh medium).  If the dilution is more than 10 fold
i.e. the DMSO is reduced to below 1% you do not even need to spin down and
wash the cells, just plate directly.  If not pellet gently, resuspend
gently and then plate as per normal.

I hope that this works for you as it does for me.

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

"Don't try and fix it if it ain't broke".
----------------------------------------------------------------------------

Newsgroups: bionet.molbio.methds-reagnts
From: wakarchu@biologysx.lan.nrc.ca (Dr. Warren Wakarchuk)
Subject: Re: X-GAL CLEAVAGE BY V. CHOLERAE
Organization: National Research Council of Canada
Date: Tue, 1 Sep 92 21:24:26 GMT

In article <1992Sep1.152134.202619@uctvax.uct.ac.za> coyne@uctvax.uct.ac.za writes:
>From: coyne@uctvax.uct.ac.za
>Subject: X-GAL CLEAVAGE BY V. CHOLERAE
>Date: 1 Sep 92 15:21:33 +0200

>Dear Netters,

>I am attempting to isolate transposon generated lacZ operon fusions in 
>Vibrio cholerae by cleavage of X-gal to generate blue colonies.  According to
>Bergeys, V. cholerae is lactose negative.  However, I have found that
>V. cholerae is able to cleave X-gal to generate blue colour which is a major 
>problem with respect to my strategy.  I would greatly appreciate it if anyone 
>could supply me with an answer to the following question:

>   Does V. choerae possesses a beta-galactosidase enzyme?
>   (The bacterium was scored as lac- as a function of growth on lactose).

>You may post the answer e-mail it directly to me.

>Many thanks in advance,

>Vernon Coyne
>vernon@micro.uct.ac.za
> 
Your problem is likely that another glycosidase like Beta-glucosidase is 
cleaving the X-gal.  Most gram negative strains have such enzymes and the 
specificity is rather "loose".  E. coli Beta-galactosidase does cleave other 
glucosides as well.  You might have to look for the enzyme itself, not its 
activity.

Warren Wakarchuk
IBS NRCC
wakarchu@biologysx.lan.nrc.ca
From: John.Hardham@bbs.oit.unc.edu (John Hardham)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Mol. Wght. prediction software
Date: 3 Sep 92 15:09:44 GMT
Distribution: na
Organization: Extended Bulletin Board Service

Does anyone out in net land know where to get a program for the Macintosh
that will predict the molecular weight of a protein based on its
mobility in a protein gel?

--
   The opinions expressed are not necessarily those of the University of
     North Carolina at Chapel Hill, the Campus Office for Information
        Technology, or the Experimental Bulletin Board Service.
           internet:  bbs.oit.unc.edu or 152.2.22.80
Newsgroups: bionet.molbio.methds-reagnts
From: gchacko@magnus.acs.ohio-state.edu (George W Chacko)
Subject: Nucleic Acid Precipitation
Organization: The Ohio State University
Date: Fri, 28 Aug 1992 18:48:40 GMT


What are your thoughts on the relative merits of

a-	isopropanol
	
b-	95 % ethanol

c-	99% ethanol

when used to precipitate RNA, DNA and synthetic oligonucleotides out
of solution? I found in ine case that 95% ethanol did not substitute
efficiently for isopropanol when precipitating RNA but I did that
experiment only once.

George
From: pnh@fcs260c2.ncifcrf.gov (Paul N Hengen)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: PBR322 - maximizing yields
Summary: Use runaway plasmids
Date: 28 Aug 92 16:11:49 GMT
Distribution: bionet
Organization: Frederick Cancer Research and Development Center

>bhjelle@carina.unm.edu () writes:

:Unfortunately, some things can only be done in pBR-based
:vectors and I have been plagued with totally miserable
:yields of one particular example of same for more than
:a month now.

I have two thoughts about your problem...

1. You may have cloned a segment of DNA that would prohibit
high copy number in any plasmid, in which case you will be
wasting much effort trying to amplify.

2. Why not spend some time subcloning into one of the
runaway plasmids that can be amplified simply by changing
temperature? This could save quite a bit of aggravation
and the bottle of chloramphenicol. See these refs...

B. E. Uhlin et al. Gene 6:91-106. (1979)
B. E. Uhlin et al. Gene 22:255-265. (1983)

Paul N. Hengen
National Cancer Institute
Frederick Cancer Research and Development Center
Frederick, Maryland 21702-1201 USA
From: BISHOP@MSVAX.MSSM.EDU ("DAVID F. BISHOP")
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Tm
Date: 26 Aug 92 23:07:31 GMT
Distribution: bionet


>Dear Netters,
>I would be grateful for any information you can give me about calculating the
>Tm's for oligo hybridization. 
>.
>.Stuff deleted
>.
>Cheers,
>George Murphy

An excellent review on Tms has recently been published:

Wetmur, James G. DNA probes:Applications of the principles of mucleic acid
hybridization. Crit. Rev. in Biochem. Mol. Biol. 26:277-259,1991.

Careful reading will reveal many important considerations for all kinds of
hybridization experiments.

David Bishop                             | EMail:Internet:Bishop@msvax.mssm.edu
Div. of Medical and Molecular Genetics   |         Bitnet:Bishop@msrcvax
Mount Sinai School of Medicine, Box 1203 | Phone: (212) 241-6946
Fifth Avenue and 100th Street            | FAX:   (212) 360-1809
New York, NY 10029                       | GEnie: D.Bishop
From: pnh@fcsparc6.ncifcrf.gov (Paul N Hengen)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: PBR322 - maximizing yields
Date: 31 Aug 92 14:40:51 GMT
Distribution: bionet
Organization: Frederick Cancer Research and Development Center

bhjelle@carina.unm.edu  writes:

:Unfortunately, some things can only be done in pBR-based
:vectors and I have been plagued with totally miserable
:yields of one particular example of same for more than
:a month now.

pnh@ncifcrf.gov writes:

>1. You may have cloned a segment of DNA that would prohibit
>high copy number in any plasmid, in which case you will be
>wasting much effort trying to amplify.

bhjelle@carina.unm.edu  writes:

>Actually the plasmid is a trpE expression construct, and I
>am having the most problems with the vector itself, rather than
>derivitives containing my inserts. The lab I got the clone from
>swear that it grows about as well as pBR, and that there is
>enough tryptophan in LB to repress the trp E anyway.
>
>I could move the whole trpE gene into pUC I suppose, but that
>would require being able to grow the pBR-based vector.

Brian: I don't see how someone could construct this plasmid
without being able to get it back from E. coli transformants.
In the past I've had problems getting plasmid DNA from cells
that just would not lyse with my standard procedure (for
reasons I can't really explain). Sometimes I've done a little
trick to get them to lyse. For example, just after the initial
resuspension in TE buffer, and before placing them in the sucrose
buffer, I've added a wash with 10 ml of 1 M NaCl, 10 min. on ice,
then centrifuge. I then resuspend the cell pellet in the usual
sucrose soln. and follow with a the lysis solutions as usual.
This seemed to help get better lysis (and plasmid yield). I've
also done other things like heat shock at 65 C for 10 min. and
then back on ice 10 min. cycled until the sup. is very viscous.
Question: Do you try the same procedure for lysis with say HB101
carrying pBR322 as a meter for plasmid yield? My feeling is that
you're just not getting thorough lysis.

Paul.
From: txt15@po.CWRU.Edu (Tao Tao)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Partial digestions with ApaL1 - help!!
Date: 1 Sep 1992 17:07:09 GMT
NNTP-Posting-Host: thor.ins.cwru.edu


For the ApaLI partial digestion, I would suggest using EtBr.  There should be
some literatures on this.  

Tao
From: wegloehner@rz-berlin.mpg.de (Wolfgang Wegloehner)
Newsgroups: bionet.molbio.methds-reagnts
Subject: MODIFIED AMINO ACIDS - HELP!!!
Date: 4 Sep 92 15:38:33 +0100
Organization: MPI fuer Molekulare Genetik, Berlin

Dear netters,

Does anybody in the net know where to get methylated amino acids? We are
especially looking for:

       N(epsilon)-trimethyl-L-Lysin   
 and   N(epsilon)-trimethyl-LAlanin

We would greatfully appreciate any suggestion concerning companies or labs.
Thanks in advance,

                         Juergen Schmidt,
               MPI fuer Molekulare Genetik, Berlin 
Newsgroups: bionet.molbio.methds-reagnts
From: bhjelle@carina.unm.edu ()
Subject: PBR322 - maximizing yields
Date: Thu, 27 Aug 92 13:24:38 GMT
Organization: University of New Mexico, Albuquerque

The postings about how to get great yields of pUC are all fine
and good, but in most cases we get enough DNA from a pUC
miniprep to keep us happy for a long time. 

Unfortunately, some things can only be done in pBR-based
vectors and I have been plagued with totally miserable
yields of one particular example of same for more than
a month now.

I have tried chloramphenicol amplification (which I used
to use in the old days when pBR was everywhere, but have
lost my old recipe). Unfortunately, the major cloning
manuals do not actually give understandable recipes for
chloramphenicol amplification. The attempts I made to
improvise did not seem to help.

What are the best tricks for increasing yields of pBR-based
vectors?

Brian

Newsgroups: bionet.molbio.methds-reagnts