HDTenEightyP/NetNews huggingface dataset:
From: JANIEMI@finabo.abo.fi (Jarmo Niemi TUY)
Subject: Re:BclI and BamHI ligation
Organization: Abo Akademi University, Finland
Date: Mon, 28 Sep 1992 13:10:49 GMT
X-News-Reader: VMS NEWS 1.23

In <9209231147.AA16613@genbank.bio.net> AWS4270%USA.decnet@USAV01.GLAXO.COM
writes:

> Call me paranoid...but EVERYTIME I alkaline phosphatased a
> vector, then gene-cleaned it, the ligations never worked.  NOTHING!
> If I purify the fragment by gene-clean, then phosphatased it and
> precipitated it by traditional means, the ligations always work!
> I don't lnow why, but it appears that phosphatased, then gene-
> cleaned vector doesn't work in my ligations.  Therefore I never
> prepare my vector that way anymore.
> 
> Anne Howe
> Glaxo
> 
In my experience CIAP, calf intestinal alkaline phosphatase is a
difficult enzyme to kill, and if any remains, it will ruin a ligation
by eating the phosphates off the inserts as well. I don't think I would
dare to use only Gene-clean. Last time, after digestion with 1 U CIAP, 
I added EDTA to 5 mM, SDS to 0.5%, incubated 40 min at 68 C, phenol
extracted, chloroform extracted and _then_ gene-cleaned. I don't know
if this is overkill, but at least it works.

Jarmo Niemi
Biochemistry, University of Turku
janiemi@finabo.abo.fi
From: ED@micro.uct.ac.za (Ed Rybicki)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Stratagene Quick-Hyb
Date: 29 Sep 92 10:38:48 GMT
Reply-To: ed@micro.uct.ac.za
Distribution: bionet
Original-To: kim@edu.unm.m44, methods@uk.ac.daresbury
X-Mailer: Pegasus Mail v2.1c R5.

> I saw an ad for Stratagene's Quick Hyb hybridization solution.  the claim is
> that hybridization of blots is reduced to 1 hour (from overnight).  Does anyone
> know what does this?  Is it just a bunch of PEG added to a regular hyb
> solution?  If so, why doesn't everyone do this?
>
> If anyone does this kind of thing regularly (i.e. 1 hour hybridizations), I'd
> like to know how.  Thanks
>
> Daniel Kim
> KIM@FLOVAX.LANL.GOV

We don't do 1-hr hybs, but we used (I don't know if the students and other
children still do, they've gone off and invented other myths since) to
include 10% PEG-6000 in hyb mixes: esp hyb mixes containing formamide and
using RNA as probe, instead of dextran sulphate.  My colleague Maud Purves
did a 2-hr hyb using a cDNA probe onto dot-blotted RNA for one of our
student practicals the other day, with 10% PEG, and it worked fine.  Like
the Biorad "negative blot" protein stain (=0.3M CuCl2) - a well-hidden
secret, much cheaper off the shelf than out of the catalogue.

Hyb on!
From: wetsel_r@wums.wustl.edu
Newsgroups: bionet.molbio.methds-reagnts
Subject: RE: immune precipitation
Date: 3 Oct 92 18:49:45 GMT
Organization: MIT PLASMA FUSION CENTER
NNTP-Posting-Host: msnews.wustl.edu

Hi Mike:

I would be very interested in any replies that you receive concerning
this subject.  I too will be metabolically labeling followed by
immunopreciptiation with SP-A.

Thanks in advance...

David
haviland@kids.wustl.edu
From: rice@embl-heidelberg.de (Peter Rice)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Umea Drosophila stocks
Date: 28 Sep 92 20:35:06 GMT
Organization: EMBL, European Molecular Biology Laboratory

I have the Drosophila stock listings from the Indiana FTP site, but our
fly labs here would like the listing from the European stock centre
at Umea in Sweden. Something to do with how long it takes a fly to fly
from the USA I suspect.

Is the Umea list available online anywhere?

 -----------------------------------------------------------------------------
 Peter Rice, EMBL                             | Post: Computer Group
                                              |       European Molecular
 Internet:    Peter.Rice@EMBL-Heidelberg.DE   |            Biology Laboratory
                                              |       Postfach 10-2209
 Phone:   +49-6221-387247                     |       W-6900 Heidelberg
 Fax:     +49-6221-387306                     |       Germany

 ... and occasionally price@crc.ac.uk ...
From: etaylor@ccu.umanitoba.ca (Euan R. Taylor)
Newsgroups: bionet.molbio.methds-reagnts
Subject: re N's with DIG
Summary: we have problems too
Date: 30 Sep 92 18:21:47 GMT
Organization: University of Manitoba, Winnipeg, Canada

We have had a similar problem, filter lifts, plasmid blots and RNA dot
blots work with a DNA probe oligolabelled with 32P. The same blots show no
obvious differences between positive and negative controls when probed
with the DIG labelled probe (same DNA probe, same filters).
We   checked the labelling of the probes by dot blotting and developing
sserial dilutions of the probe onto a membrane alongside the supplied +ve
control probe, and got fine signals, if anything slightly better than the
control. We did the blots, got development but everything looked like the
negative control.(nb. plasmid blots-lots of DNA, benton davies lifts, both
giving extremely strong clear +ves with the 32P).
So we ran a gel of the probes on PACM, blotted and developed it, probe
fragments are well up above 100-200 bp. not that much different in
appearance from the supplied control probe.
so then we did one more expt. re spotting our probes and the boehringer probe
this time we cant see our probes coming up at all whilst we can see the
ssupplied control.
We tried a series of UV exposures for fixing the DNA to the filters- this
made no apparent difference to the absolute or relative signal strengths.

So- when we run it on a gel the probe seems to be fine, when we blot it in
a dot blot -it ain't.

We have beeen storing all the probes side by side in the same box, in  the
appropriate dilution buffer, at -20C. But I'm beginning to wonder if there
might be some stability poblem nevertheless. any ideas?

NB. People down the road are doing N'ns with DIG RNA and getting great
results. Buggered if I know what's going on.

Euan Taylor
Dept. Plant Science
U of Manitoba
From: kim@m44.unm.edu
Newsgroups: bionet.molbio.methds-reagnts
Subject: Stratagene Quick-Hyb
Date: 28 Sep 92 18:18:26 GMT
Reply-To: kim@m44.unm.edu
Organization: UNM Cancer Center, Albuquerque, NM

Helllo Group:

I saw an ad for Stratagene's Quick Hyb hybridization solution.  the claim is
that hybridization of blots is reduced to 1 hour (from overnight).  Does anyone
know what does this?  Is it just a bunch of PEG added to a regular hyb
solution?  If so, why doesn't everyone do this?

If anyone does this kind of thing regularly (i.e. 1 hour hybridizations), I'd
like to know how.  Thanks

Daniel Kim
KIM@FLOVAX.LANL.GOV
From: CZJ@CU.NIH.GOV
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re:  pH problem
Date: 5 Oct 92 13:05:13 GMT
Distribution: bionet

>
> I am having problems adjusting the pH of various 1 M solutions (citrate,
> succinate etc.). My pH electrode, if left in these for a while (few minutes)
> seems to drift, in the sense that when I rinse it with distilled water, and put
> it back into my reference buffer, I get a lower pH value than I know it should
> be. This obviously doesn't bode well for accurate pH's in the above mentioned
> solutions! What exactly is the influence of ionic strength on pH? I also don't
> always get corresponding pH values with pH-paper. Can anyone tell me what is
> happening to the electrode?
>
> Regards,
> Pierre Janssens
> Zoology Dept.
> University of Cape Town
>
>

The influence of ionic strength on pH is fairly straight
forward.  pH is defined as -log activity hydrogen activity.
The relationahip between hydrogen ion activity and hydrogen
ion concentration can be calculated from the Debye Huckel equation,
although the approximation implicit in the equation begins to fall
apart at high ionic strengths, so be sure to use the extended form.

Drifting--If your solutions are not buffered and made from distilled
water, you could be picking up CO2.  Or else perhaps you have
a bad glass electrode.  Why it should not return to the
proper pH using a reference buffer suggests that.  Also check
the reference electrode.  Perhaps the porous plug is clogged.

Hope this helps

Jim Cassatt
From: skspoidn@susssys1.reading.ac.uk
Newsgroups: bionet.molbio.methds-reagnts
Subject: immune precipitation
Date: 1 Oct 92 16:45:49 GMT
Distribution: bionet
Original-To: methods@uk.ac.daresbury

I am trying to Immune precipitate a cellular protein 
(CD4) with Mabs using protein A and S35 metabolically 
labelled cells, and finding that the immune reaction is
 not occuring, although antibodies are being precipitated.
 Is there some aspect of the immune reaction and susequent
 precipitation which is critical in this procedure?

Thanks in advance

Mike poidinger
Dept of Microbiology
University of Reading
Newsgroups: bionet.molbio.methds-reagnts
From: broe@aardvark.ucs.uoknor.edu (Bruce Roe)
Subject: Re: Cycle Sequencing Problems
Originator: news@midway.ecn.uoknor.edu
Date: Sat, 3 Oct 1992 12:24:00 GMT
News-Software: VAX/VMS VNEWS 1.41    
Organization: University of Oklahoma - University Computing Services

In article <1992Sep14.190403.7540@ac.dal.ca>, batc@ac.dal.ca writes...
> 
>  Hi dear netters
>  I have figured out that there are some people who are working on cycle 
>sequencing. I 've tried some times and I had had one constant problem.
>Though I 've got really strong signal I 've got stops in all four lanes.
>I was trying to sequence PCR products using (end labeled) one of the PCR primers
>and the other was internal primer. I hane used the New England Biolabs'
>"CircumVent Thermal Cycle Sequencing Kit". Is there any one who has any idea
>why this happens and what the solution could be? I would appreciate any
>oppinion.
>Thank you in advance
>Costas Batargias

Hi,
	Your problems could be due to several factors:
1. Garbage template - try cycle sequencing off highly purified pUC or
			some other very good control DNA.
2. Letting the reactions sit at room temperature too long either before
	or after cycling.  One of my students recently obtained multiple
	stops in all 4 lanes similar to what you have just described and
	it turns out that he was pipetting with his tubes in a rack on the
	bench rather than in an ice/water bath, then waiting upwards to
	5 minutes before putting them in the PECetus9600.  After the
	cycling reactions were done he then had an additional 5-10
	minutes at room temp on the bench before pooling and EtOH pptn.
	The combination of all this time at room temp. produced gels
	with lots of "template-associated" compression due to the Taq
	polymerase activity which DOES CATALYZE REPLICATION, abite slowly,
	even at room temperature.  But the template's secondary structure
	is not melted out and thus the "template-associated" compression.
	SUGGESTION: to be extra carefull, pipet the cycle sequencing reactions
		    on ice (ice/water bath) and pool the reactions after
		    placing them back into the ice/water bath once cycling
		    is complete.  Bottom line is don't let the reactions
		    sit at room temperature either before or after cycling
		    as the enzymes do have activity at these lower temp's,
		    just enough to screw things up........sigh
	At the recent Hilton Head Human Genome meeting, Bill Studier
	described a new 6-mer based primer sequencing method that had
	as it's initial step a primer extension at aprox. 0 deg C.
	i.e. in an ice/water bath.........gives one pause to think...
Cheers....bruce
  - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
 \  Bruce A. Roe                     Dept. Chemistry and Biochemistry /
 /  BROE@aardvark.ucs.uoknor.edu     University of Oklahoma           \
  - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
From: batc@ac.dal.ca
Newsgroups: bionet.molbio.methds-reagnts
Subject: Messages are received. Thanks a lot
Date: 26 Sep 92 20:48:41 GMT
Organization: Dalhousie University, Halifax, Nova Scotia, Canada

Hi netters
I was really delighted receiving 17 or so messages answering either to my 
message for help or to the other for cycle seq. 
Thank you very-very much for your responses on both subjects.
Costas Batargias
From: sbhattac@crc.ac.uk (Dr. Shoumo Bhattacharya B1 061)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Looking for a good oligo
Date: 28 Sep 92 15:02:53 GMT
Distribution: bionet
Original-To: methods@uk.ac.daresbury

I have used the Primer program (from Eric Lander) with good results.  It
is easy to use, and above all, is FREE.  I can recommend it.
Shoumo
From: djt2@po.CWRU.Edu (Dennis J. Templeton)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Can anyone help with protein expression in pGEX vectors?
Date: 6 Oct 92 21:34:51 GMT
Reply-To: djt2@po.CWRU.Edu (Dennis J. Templeton)
Organization: Case Western Reserve University, Cleveland, OH (USA)
NNTP-Posting-Host: thor.ins.cwru.edu


In a previous article, lhanson@moose.uvm.edu (lhanson) says:

>	I've been trying to express 2 cDNAs in pGEX-2T.  Both cDNAs are

>(Deleted sad but all too typical tale of poor expression or solubility of 
>(fusion proteins

We have had in one case a near-miraculous improvement in solubility by
changing the temperature of the induced culture.  At 37 degrees it was
about 5% soluble, at 30 degrees it was more than 50% soluble, and the
expression was the same.  25degrees was slightly better.  For this one
protein, our routine is now to grow uninduced to about O.D. 0.5, then
induce and shut off the heat, then shake overnight.  

In other cases of poor solubility, this trick has worked *a little*...

It seems worth a try...

good luck,

dennis
From: mills_j@mscf.med.upenn.edu
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: whence Coomassie?
Date: 5 Oct 92 03:41:28 GMT
Distribution: bionet,world
Organization: Univ of Penn - Med School Computer Facility

BI1RGW@ibm.shef.ac.uk writes:
> Hello Netland.
> 
> Can anyone remind me of the details of what, how and when Coomassie R/G bind
> to proteins?  As I recall, R binds slower but is more sensitive - but what do
> they bind?  I have at the back of my mind some vague inkling about lysine
> residues - but what about arginine, and what about the N-terminus?
> 
> I'm sure this is a FAQ, but...all comments, recollections, anecdotes welcome.

    You know, I spent a little bit of time researching just what exactly Coo-
massie dye binds to.  The basic conclusion that I reached at the time was 
that either nobody knows, or I was looking in the wrong places.  There is a
whole chapter on various anionic dyes in a 1985 book on electrophoresis, but
as I remember it, other than a lot of interesting stuff about the history and
some explanations of sensitivity and a good bit of detail about linearity of
staining intensity to mass of protein, there wasn't much about binding.  All
I remember was that Coomassie was supposed to bind free amino groups.  Of
course, that would dictate lysine binding, but I got the impression the in-
teraction between dye and protein wasn't that specific.

    I'd be a little surprised if this were a FAQ, but if it is, or if there 
are any dye organic chemists out there chomping at the bit to enlighten this
cell biologist who's out of his league, I'd be interested.
-- 
                                  JCM                      
             |---------------------------------------------|
             |--Mills_J@a1.mscf.upenn.edu (Jason Mills)----|
             |"Why would anyone want both an MD and a PhD?"|
             -----------------------------------------------
From: kaya@mscf.med.upenn.edu
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Photographing Coomasie-stained Gels
Date: 29 Sep 92 21:52:34 GMT
Organization: Univ of Penn - Med School Computer Facility

I photograph with a light box and prefer a yellow filter for Coomassie Blue 
stained gels.

Sorry, I only take Polaroid Type 667 film (no negatives).

Hope this helps.

angie.
From: afc@gnv.ifas.ufl.edu
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: FAQ!!
Date: 1 Oct 92 11:26:53 -0500
Followup-To: chloramphenicol [faq]
Distribution: bionet

In article <4001@fcs280s.ncifcrf.gov>, pnh@fcsparc6.ncifcrf.gov (Paul N Hengen) writes:
> 
> Due to the recurrence of the chloramphenicol question,
> I've started the construction of a FAQ list for
> bionet.methds-reagnts and would like other people to
> contribute to it's growth and evolution. Please feel free
> to include what you like and any suggestions for a better
> FAQ list. Thank You. -Paul.

I think a FAQ would be useful.  My suggestion would be to leave the
protocols out- there are too many to consider.  This is especially
true of ones like plasmid amplification, where a good reference exists.

I would suggest that a list of definitive papers would be great, e.g.
"read Hanahan for questions on transformation of E. coli".  We could
come up with similar "read this before posting" papers on DNA isolation,
hybridization, etc.  Techniques like PCR that are still largely black
magic probably haven't been adequately reviewed yet.

Second, a list of lab manuals (Maniatis, Berger/Kimmel, etc) with a 
BRIEF list of techniques covered would also be helpful.  For example, 
if my sequencing reactions aren't working, does Maniatis discuss 
sequencing?  Actual reviews are probably not a good idea because of 
legal problems and personal preferences...

Finally, people are getting the idea that they should post summaries of
answers when they ask a question.  Is anyone archiving these?  If so,
an index and FTP address would be wonderful.

Just my $0.02 worth.

Andrew Cockburn
USDA
Newsgroups: bionet.molbio.methds-reagnts
From: saparici@crc.ac.uk (Dr. S.A.J.R. Aparicio)
Subject: Re: Size of exons and introns?
Reply-To: saparici@crc.ac.uk (Dr. S.A.J.R. Aparicio)
Organization: University of Cambridge, Department of Medicine
Date: Wed, 7 Oct 1992 00:57:48 GMT


The smallest introns I have seen published were recently shown in an article
describing the sequence of Paramecium beta-tubulin. Two 27bp introns. Paramecium
isn't human though! The shortest I think I've seen in human was 68bp.

As for the longest ... Have a look at Dystrophin, some these introns are 100Kb plus
or Dopamine D2 receptor, exon 1 is 250Kb.


regards to all


Sam Aparicio
MGU Department of Medicine
CAmbridge UK
Newsgroups: bionet.molbio.methds-reagnts
From: sknapp@helix.OSCS.ORST.EDU (Steven J. Knapp)
Subject: p33
Reply-To: sknapp@helix.OSCS.ORST.EDU
Organization: University Computing Services - Oregon State University
Date: Wed, 30 Sep 1992 16:59:41 GMT

Does anyone out there have experience with p33 relative to ChemiLum
and p32?  We would like to try p33 if it is worth the money.

Willy
From: djt2@po.CWRU.Edu (Dennis J. Templeton)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: immune precipitation
Date: 2 Oct 92 21:16:09 GMT
Reply-To: djt2@po.CWRU.Edu (Dennis J. Templeton)
Organization: Case Western Reserve University, Cleveland, OH (USA)
NNTP-Posting-Host: thor.ins.cwru.edu


In a previous article, skspoidn@susssys1.reading.ac.uk () says:

>I am trying to Immune precipitate a cellular protein 
>(CD4) with Mabs using protein A and S35 metabolically 
>labelled cells, and finding that the immune reaction is
> not occuring, although antibodies are being precipitated.
> Is there some aspect of the immune reaction and susequent
> precipitation which is critical in this procedure?
>
>
You should be aware that some, even most, monoclonals don't bind directly
to PrA, I'd cite the classes here but I'd probably mix them up.  We usually
test a new mAb empirically by binding some to PrASepharose and eluting and
staining for the IgG on a PAGE.

If your mAb doesn't bind to PrA directly, not all is lost, as you can add
rabbit anti-mouse IgG to sandwich the mAb to the PrA with the rabbit IgG
that binds pretty efficiently.  In my experience, the Rabbit anti-mouse
serum doesn't affect the quality of the IP's very much.

good luck, 

dennis
From: eric@bcserv.wustl.edu (Hugo (Eric))
Newsgroups: bionet.molbio.methds-reagnts
Subject: TCP-1 clones sought
Date: 6 Oct 92 13:11:05 GMT
Organization: Washington University in Saint Louis, MO USA
Distribution: na
NNTP-Posting-Host: bcserv.wustl.edu
Summary: Does anyone have clones of TCP-1?
Keywords: TCP-1 hsp60 yeast mammalian

Dear Netters,
     I'm searching for a cDNA clone of the yeast or mammalian TCP-1
protein (HSP60).  If anyone has a clone that they would be willing
to release or knows of a lab that can provide me with some, if you would
please email me I would appreciate it.  Thank you.
E
-- 
  Eric R. Hugo, Postdoctoral Research Associate |eric@bcserv.wustl.edu
  Dept. of Biochemistry and Molecular Biophysics|                
  Washington University School of Medicine      |               
  Box 8231, St. Louis, MO 63110_________________| (314) 362-3342
Newsgroups: bionet.molbio.methds-reagnts
From: naffara@crc.ac.uk (Dr. N.A. Affara)
Subject: Size of exons and introns?
Organization: MRC Human Genome Resource Centre
Date: Wed, 30 Sep 1992 14:51:14 GMT

This may have been asked before....

Does anyone know the maximum, minimum, (if any) and average size of exons
and introns in human DNA?

The reason I am asking is that I am trying to design PCR primers from 
cDNA sequence and I want to have some idea of the exon/intron sizes.

Thanks,
Phil
(E-mail naffara@uk.ac.crc)

-- 
Kryten (When Cat says why don't we raise the defensive shields?) "A superlative
suggestion sir,with only two drawbacks: one,we don't have any defensive shields
and two,we don't have any defensive shields.I know that,technically,that's only
one drawback,but it was such a big one I thought I'd mention it twice".
From: tolvanen@klaava.Helsinki.FI (Martti Tolvanen)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Sequence of Bluescript
Date: 29 Sep 92 14:03:44 GMT
Organization: University of Helsinki

This must be a FAQ, but I'll ask it once more: Is the sequence of the
Bluescript (TM) cloning vector available somewhere? If not, does someone
have an up-to-date restriction map? The Stratagene info sheet does not
cover the "latest" enzymes. 

-- 
Martti Tolvanen, Dept. Biochem., Univ. Helsinki, Finland
tolvanen@cc.helsinki.fi
From: wetsel_r@wums.wustl.edu
Newsgroups: bionet.molbio.methds-reagnts
Subject: RE: PCR enhancers
Date: 30 SEP 92 11:36:16 GMT
Organization: MIT PLASMA FUSION CENTER
NNTP-Posting-Host: msnews.wustl.edu

Hi Bob:

Quick question:  Who makes Hot Start? Or is that a name for a "home-
grown" recipe????

David
haviland@kids.wust.edu