HDTenEightyP/NetNews huggingface dataset:
From: num208ww@MBDS.NRC.CA (Warren Wakarchuk)
Subject: Re: Problem isolating M13 ssDNA
Reply-To: num208ww@MBDS.NRC.CA
Organization: NRC, OTTAWA, ONTARIO, CANADA.
Date: Fri, 28 Aug 92 00:47:02 GMT

In article <24AUG199200290632@vaxa.weeg.uiowa.edu>, agoodrid@vaxa.weeg.uiowa.edu (Stephen Klautky) writes:
>Usually, I work with dsDNA but on occasion I make ssDNA to
>sequence troublesome clones.  
>
>Currently, I am having some trouble with my yields.  The problem
>may be at the phage denaturing step but I'm not sure.  I'm using
>DH5-alpha-F' cells, btw.

>>stuff deleted
>
>P.S. the phage sups infect DH5-alpha-F' cells to make plaques on
>YT plates so I don't think that's the problem.
>
>Any info by posting or e-mail is appreciated.
>
>- Steve Klautky -     AGOODRID@VAXA.WEEG.UIOWA.EDU
>
I'd like to offer a couple of explanations and hints about low yields
for ssDNA preps.
1) In my hands DH5-alpha-F' is a low yield producer of ssDNA, I've had
much better luck with MV1190 (or even JM101 in the olden days).  If
you check the titre of phage you'll probably see only 1 E+10 per ml,
which is quite low compared to the 1 E+12 you can get with other
strains.  You may need to work up a 10 ml prep just to get enough for
a couple of reactions!
2) Your "troublesome clones" may be resonsible for your decreased
yields, many clones simply do not produce ssDNA well. Try a DIGE
(direct in gel electrophoresis) to see if your sup's worth working up.
3) The PEG precipitation method works well, but you have to work to
resuspend the pellets before phenol extracting them, other wise you
lose everything.  Let the pellets sit at RT for 5 min. in TE then resuspend
them, they go into solution much better. I use a glass/NaOCl4 method
now which is much faster and gives twice the yields of phenol
extraction (It is not glass milk but glass fibre filter, and I think
it is a great method).
Hope these tidbits are usefull.
*************************************************************************
Dr. Warren Wakarchuk   Prefered email: wakarchu@biologysx.lan.nrc.ca
                       Alternate     : NUM208WW@MBDS.NRC.CA
National Research Council Canada, Protein Structure and Design
Ottawa, Ont., Canada K1A 0R6 ***THESE ARE MY OPINIONS NOT THOSE OF NRC***
Newsgroups: bionet.molbio.methds-reagnts
From: djt2@po.CWRU.Edu (Dennis J. Templeton)
Subject: Looking for hGH to quantify expression from transfections
Reply-To: djt2@po.CWRU.Edu (Dennis J. Templeton)
Organization: Case Western Reserve University, Cleveland, OH (USA)
Date: Thu, 27 Aug 92 22:21:55 GMT


I've seen folks who include a plasmid that expresses human growth hormone
in co-transfections when doing CAT-style assays, then normalize for
transfection efficiency after assaying for hGH by ELISA (I think)

What is the source for the hGH expression clone, and the assay?

Might anyone have a vector they can share?

thanks,

dennis
From: MURPHY@UK.AC.AFRC.JII
Newsgroups: bionet.molbio.methds-reagnts
Subject: TM
Date: 26 Aug 92 12:13:00 GMT
Distribution: bionet
Original-To: METHODS@UK.AC.DARESBURY

Dear Netters,
I would be grateful for any information you can give me about calculating the
Tm's for oligo hybridization. My problem is this:
Sambrook/Fritsch/Maniatis (whose endeavours I have been grateful for on many
occasions) quote three equations to calculate Tm's (Vol2, p9.51). As has been 
pointed out before, the value for log[Na] should be added, not subtracted in
the DNA/DNA equation. Nobodys perfect (:^). My interest is in the DNA/RNA and 
RNA/RNA equations.
1. Where %G+C is mentioned, then I assume 60%G+C is 0.6 and not 60 (which 
isn't %G+C in my book)?
2. Can anybody provide me with references for these equations? I have looked
at the ones quoted in Sambrook et al. but I cannot find the equations in these
papers. Thanks in advance.
Cheers,
George Murphy
********************************************************************************
George Murphy                     |Snailmail: 
                                  |     Molecular Genetics,
JANET:murphy@uk.ac.afrc.jii       |     Cambridge Laboratory,
INTERNET:murphy@jii.afrc.ac.uk    |     JI Centre for Plant Science Research,
                                  |     Colney Lane,
Telephone: +44 603 52571          |     Norwich NR4 7UJ,
Fax:       +44 603 505725         |     U.K.
********************************************************************************
Newsgroups: bionet.molbio.methds-reagnts
From: Bo Servenius <biogen10@gemini.ldc.lu.se>
Subject: Solid phase sequencing of long inserts
Organization: Lund University, SWEDEN
X-Useragent: Nuntius v1.1
Date: Mon, 31 Aug 1992 22:23:42 GMT

Dear Netters:

Planning to do some solid phase sequencing with the kit from
ABI. To my surprise I see theat they do not recommend you to use
it for fragments longer than 1kb - due to poor binding to the
Dynabeads. 

I would very much appreciate any commnts from anyone that has
tried longer fragments.

Yours

BOSSE
Newsgroups: bionet.molbio.methds-reagnts
From: zxmkr08@mailserv.zdv.uni-tuebingen.de (Cornelius Krasel)
Subject: Protein-protein interactions: summary
Summary: (see subject)
Keywords: Far western blot, expression cloning, affinity chromatography
Organization: Comp. Center (ZDV) U of Tuebingen, FRG
Date: Thu, 3 Sep 1992 06:23:26 GMT


Dear bio-netters,

a few weeks ago, I asked for methods to study protein-protein interactions, and
promised to summarize. Below are the answers which I received by email. I
would like to add that I found an article in TIBS 17(1992),246 by Daniel
W. Carr and John D. Scott titled "Blotting and band-shifting: techniques
for studying protein-protein interactions". Another suggestion which was made
on the net was to construct an expression library in yeast where the ORFs
are fused to a part of GAL4 protein (references are Fields and Song, Nature
340(1989),245-246 and Chien et al., PNAS 88(1991),8578-9582).

Thank you for your responses,
--Cornelius.

~From: NANTEL@UNCVX1.BITNET

I personnaly know (and use) two other techniques for studying protein-
protein interactions on top of crosslinking and immunoprecipitation:

Far western blot: Similar to a western blot except that a labelled protein
is used as a probe. The probe can be biotinylated or labelled with 32P,
125I or 35S. The following references will be useful:
        Oncogene (1990), 5, 451-458
        Mol Endocrinol (1991), 5, 256-266
        Genes & Dev (1992), 6, 439-453
        Science (1992), 257, 803-805

Affinity chromatography: Express one of your protein as a GST (or MBP)
fusion in E. coli. These fusion proteins will then bind to gluthatione
sepharose or amylose resins and can be used to fish out binding proteins.
Numerous labs have used this technique which was originally described by
Kaelin et al. in Cell (1991), 64, 521-532.

Andre Nantel
Dept. of biology
University of North Carolina-Chapel Hill
"Murphy's law is strong in molecular biology"

~From: ccy@po.CWRU.Edu (Cheung C. Yue)

Another method is expression cloning.  An expression library is probed
with a labelled protein (or peptide domain of a protein) to search for
cDNA clones expressing an epitope which binds/interacts with the protein
probe.  That way, you get the gene for the interacting protein; thereby
saving enormous efforts in protein isolation, purification and sequencing.
A number of successful applications have appeared in the literature in 
the recent past.

ccy@po.cwru.edu
--
/* Cornelius Krasel, Department of Physiological Chemistry, U Tuebingen    */ 
/* email: krasel@mailserv.zdv.uni-tuebingen.de (Internet)                  */
/*        krasel@chemie.uni-tuebingen.dbp.de (WIN/X400)                    */
/* "People are DNA's way of making more DNA." (anonymous)                  */
From: BAKERK@UK.AC.AFRC.FRIR
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Sf9 Cells
Date: 29 Aug 92 13:54:00 GMT
Distribution: bionet
Original-To: METHODS@UK.AC.DARESBURY

>  Hi! I was wondering if anyone out there has experience working with Sf9 cells
>isolated from the ovary of armyworms.
>  I pretty much follow the protocol of Summers and Smith. Using Grace's medium
>supplemented with 10% FCS and the specified concentration of Gentamicin.
>  However, recently my cells begin to die after a few days of incubation.
>Specifically, after reviving the cells from liquid N2 and seeding 2e6 cells in
>a 25cm2 flask, the cells are fine and reaches confluency. I would then pass the
>cells 1 to 3 or 1 to 4 in about 5 days. But appoximately 2 days after the 1st
>passage, the cells would begin to die. They would float, stop dividing, and I
>think that I can see holes in them (some bright spots?). Don't think it's
>contamination by baculovirus though that's what I'm growing the cells for?
>Has anyone experienced this? Any suggestions?
>  Are these cells susceptible to mycoplasma contamination? The gentamicin that
>I'm using is from BRL and the maker describes as effective against mycoplasma.
>  I've also heard that a "new?" medium called Sf900 is better. Any comments?
>   How many passages can I achieve with these cells continously
>  Right now, I plan on purchasing new cells from INV.
>  I would appreciate any comments or suggestions.
>  Thanks in advance.

>a52041@sakura.kudpc.kyoto-u.ac.jp
>Michael Cheng

The 'holes' that you can see are almost definitely polyhedra, indicating that 
your cells are infected with polyhedrin-positive baculovirus. Throw out all of 
your opened bottles of medium, serum, etc., buy or thaw out new cells and start 
again.
Better luck next time.
Cheers,

Ken.
*************************************************************************
Dr Ken Baker                              JANET : UK.AC.AFRC.FRIR::BAKERK
Department of Protein Engineering      INTERNET :  BAKERK@FRIR.AFRC.AC.UK
AFRC Institute of Food Research             TEL :        (+44) 734 357139
Reading                                     FAX :        (+44) 734 267917
Berks RG6 2EF
UK                          (AFRC = Agricultural & Food Research Council)
*************************************************************************
From: drintoul@matt.ksu.ksu.edu (David A Rintoul)
Newsgroups: bionet.molbio.methds-reagnts
Subject: hydrolysis of hydrophobic proteins
Date: 3 Sep 1992 08:34:26 -0500
Organization: Kansas State University
NNTP-Posting-Host: matt.ksu.ksu.edu

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 that
is giving us trouble.  The protein is extremely hydrophobic; we
isolate and purify it by extracting the membranes with chloroform and
methanol (1/1) and run the extract over a lipophilic Sephadex column.
By SDS-PAGE, is is pure (no other self-respecting protein would hang
around for that kind of treatment!).  Now we need to do an amino acid
analysis to quantitate the stuff, and to ensure that it corresponds to
the amino acid composition predicted from the cDNA sequence.

The approaches thus far have been to dry it down under nitrogen,
hydrolyze it in HCL, and assay the amino acids by HPLC (pico-tag or
ninhydrin absorbance).  We tried hydrolyzing it for 24 hours, and the
analysis indicated a substantial amount of something which was vaguely
identified as unhydrolyzed protein.  The amount of amino acid released
was also consistent with this diagnosis; it was about 40% of what we
expected.  So, we tried hydrolyzing it for 72 hours, and the results
are the same (even worse).

Several other facts need to be considered as well.  When we run the
protein on SDS-PAGE, we never boil it, since others have shown (and we
have corroborated) that it irreversibly aggregates at this
temperature.  Could the temperature that we use for hydrolysis (105 C)
be the problem?  If so, how do you hydrolyze proteins quantitatively
without heating them?  The other fact is that after drying the protein
down from the solvent (1/1 chloroform/methanol), it cannot be
resuspended in solvent, indicating that is is also irreversibly
aggregated and insoluble in any solvent (chloroform, methanol,
mixtures of these, ethanol, trifluoroethanol etc).  

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
Biology Division - KSU     Latitude 39.18, Longitude -96.34
Manhattan KS 66506-4901                         TCN: id2418
(913)-532-5832 or 6663                  FAX: (913)-532-6653
From: byrne@soma.umdnj.edu (Bruce Byrne)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: PCR with E. Coli
Date: 4 Sep 92 18:07:56 GMT
Distribution: bionet

ANSIDHP@EARN.OSUCC writes:
[stuff deleted]
>I am now about to help a colleague attempt to amplify a large
>(%2 kb) fragment in E. Coli.
>For mammalian DNA I normally use:
>100 ng genomic template
>300 nM each primer
>200 uM each dNTP
>1.5 mM MgCl
>in 50 ul reactions.
>Should I alter any of these conditions for amplification in
>E. Coli? 
[more stuff deleted]


Reduce DNA concentration by 10^3 to get same number of targets.  
High concentration of targets will kill your reaction.
Concentration of primers:targets will remain constant as
you are used to.  Your Mg concentration will need to be 
optimized to primer sequence.  Watch out for EDTA in DNA buffers.
Your Mg is at the low end of my experience.  Your 1min x 3 cycle
is probably a sufficient extension time and longer than necessary
for denaturation or annealing.  You may be cooking some taq
unnecessarily.

>Any helpful comments would be greatly appreciated!
     ^^^^^^^
     ...if it works, it was helpful.  Good luck.

Bruce Byrne


>Daniel Pomp
>Department of Animal Science
>Oklahoma State University
Newsgroups: bionet.molbio.methds-reagnts
From: djt2@po.CWRU.Edu (Dennis J. Templeton)
Subject: Re: Tacking down cells
Reply-To: djt2@po.CWRU.Edu (Dennis J. Templeton)
Organization: Case Western Reserve University, Cleveland, OH (USA)
Date: Sat, 29 Aug 92 17:12:02 GMT


In a previous article, BMA14432@VAX1.UTULSA.EDU () says:

>
>Hello netters,
>I recently read somewhere about a product that will "tack down"
>non adherant cell to a dish or plate. Does anyone know of this product. Where 

A product named "cell tack"(?) used to be (4-5 years ago) advertised in the
back pages of the popular journals.  It was essentially barnacle glue, and
was most useful to leukocyte biologists, I think.  I can't recall the
company name, and I havent seen it advertised recently.  Any more help out
there?
From: axa12@po.CWRU.Edu (Ashok Aiyar)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Partial digestions with ApaL1 - help!!
Date: 1 Sep 1992 15:42:57 GMT
Organization: Case Western Reserve University, Cleveland, OH (USA)
Reply-To: axa12@po.CWRU.Edu (Ashok Aiyar)
NNTP-Posting-Host: thor.ins.cwru.edu


I'm doing partial digestions on a plasmid that has 4 ApaL1
sites, and can't get the partials to work.  I have tried to
dilute the enzyme over 2 logs and independently have also tried
timed digestions.  I seem to get the enzyme cutting at all four
sites or none of the sites.

Please help!

Ashok
-- 
                          Ashok Aiyar
   		       axa12@po.cwru.edu
                Visit the IBM-PC Sig on Freenet
From: dknudson@ops.agsci.colostate.edu
Newsgroups: bionet.molbio.methds-reagnts
Subject: Followup on Tm references
Date: 26 Aug 92 14:33:54 GMT
Reply-To: dknudson@ops.agsci.colostate.edu ()
Organization: Colorado State University, College of Agriculture


I hasten to add to my reply to Murphy's query that the formulae references were
for long molecules.  The references for short are:
Suggs etal ICN-UCLA Symp. Dev. Biol. using Purified Genes Vol 23, pp683-693(81)
Rychlik&Rhaods NAR 17:8543-8551(89).
From: micprf@lure.latrobe.edu.au
Newsgroups: bionet.molbio.methds-reagnts
Subject: what is ibz0?
Date: 27 Aug 92 15:36:49 GMT
Organization: VAX Cluster, Computer Centre, La Trobe University

Hello molbio freaks,
	A colleague has asked me to ask you about the restriction
enzyme IBz0. What type of restriction enzyme is it, where does it cut,
where can one get hold of it etc? Please send replies to
fisher@mpib-martinsried.mpg.dbp.de
as I am posting this to the net via my account in Australia. We
dont have access to news here in Martinsried in Germany. Thanks,
							Paul Fisher.
Newsgroups: bionet.molbio.methds-reagnts
From: bchs1b@Elroy.UH.EDU (Michael Benedik)
Subject: Re: PBR322 - maximizing yields
Reply-To: bchs1b@Elroy.UH.EDU
Organization: University of Houston
Date: Thu, 27 Aug 1992 15:48:58 GMT

In article <wpcnbyr@lynx.unm.edu>, bhjelle@carina.unm.edu () writes:
>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
>

the general recipe for chloramphenicol amplification is to add chloramphenicol
at a concentration of 50-100 microgram/ml. The precise concentration is
not that important. It is usually added when the culture reaches
a density of between 0.5-1.0 OD (A-600). Allow amplification to continue
for 12-18 hours. 

	---------------------------------------------------
	 Michael Benedik
	 Department of Biochemical and Biophysical Sciences
	 University of Houston
	 INTERNET: Benedik@UH.EDU	BITNET: Benedik@UHOU
From: wmelchior@NTBTOX.NCTR.FDA.GOV ("Bill Melchior, NCTR/FDA")
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Modified Amino Acids - help
Date: 4 Sep 92 19:37:37 GMT
Reply-To: wmelchior@NTDOC.NCTR.FDA.GOV
Distribution: bionet

>       N(epsilon)-trimethyl-L-Lysin   

Available from Sigma.
From: bchs1b@Elroy.UH.EDU (Michael Benedik)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: PBR322 - maximizing yields
Date: 3 Sep 92 14:56:14 GMT
Reply-To: bchs1b@Elroy.UH.EDU
Distribution: bionet
Organization: University of Houston

In article <a1dnm1#@lynx.unm.edu>, bhjelle@carina.unm.edu writes:
>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

Amplification should certainly help. But another suggestion is to try
a different E.coli strain. I certainly find that I get higher plasmid
yields in recA+ strains than in recA-, so for example if you are in
HB101 or DH series you might want to try something else. 
Have you had trouble getting good yields with other plasmids. The problem
may be something other than this plasmid, just that with pUC vectors even
a bad yield still gives plenty of DNA. You might look over your plasmid
prep reagents etc.

	---------------------------------------------------
	 Michael Benedik
	 Department of Biochemical and Biophysical Sciences
	 University of Houston
	 INTERNET: Benedik@UH.EDU	BITNET: Benedik@UHOU
From: lonetto@psl.wisc.edu (Michael Lonetto)
Subject: Need E.coli vector w/T7 and regulated promoters
Reply-To: lonetto@psl.wisc.edu
Organization: University of Wisconsin - Physical Science Lab
Date: Mon, 31 Aug 92 16:50:48 GMT


Hi,

I'm looking for a vector with both a regulated (E. coli) promoter and
a T7 promoter for overexpression of the same product.  Before I go
ahead and make it I'd like to know if someone else has already done
it and is willing to give it out. The commercial vectors I've seen with
P-lac and P-T7 all seem to produce fusions to B-gal or a T7 protein.  
For my purposes I need to express my gene as a non-fusion under regulated
conditions (for study IN VIVO), as well as overexpress it (for purification).

This seems like something that a lot of people working in E. coli would
use, so I'd be surprised if no one had done it yet.

Thanks,

Mike Lonetto
lonetto@psl.wisc.edu

Michael Lonetto       UW Bacteriology  Madison, WI 53706   (608)262-4828
lonetto@psl.wisc.edu   lonetto@calshp.cals.wisc.edu
From: moseley@u.washington.edu (Steve L. Moseley)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: PBR322 - maximizing yields
Date: 28 Aug 92 17:09:21 GMT
Organization: Microbial Pathogenesis and Motorcycle Maintenance


My experience with chloramphenicol amplification was that the earlier in log 
phase you add the chloramphenicol, the better. I recall adding it at about 
0.5 OD 540 for the best yields. Also, I seemed to get much improved yields 
with normal aeration. Contrary to folklore, the fragility of the cells didn'
t seem to be a problem.
__________________________________________________________________________
Steve L. Moseley                                  moseley@u.washington.edu
Microbiology SC-42                                Phone: (206) 543-2820
University of Washington                          FAX:   (206) 543-8297
Seattle, WA 98195