HDTenEightyP/NetNews huggingface dataset:
From: Klaus.Matthaei@anu.edu.au
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Oligos for sequencing...
Date: 11 Nov 92 22:11:21 GMT
Distribution: bionet

>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

G'Day

After deprotection we simply dry our oligos down on a speedvac, wash once
with about 100ul of water, dry down again and dissolve in water.  These can
be directly used in PCR, sequencing etc. after quantitation by OD readings
at 260.

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

"If all else fails : Read the instructions"
--------------------------------------------------------------------------

Newsgroups: bionet.molbio.methds-reagnts
From: asweatma@crc.ac.uk (A.K. Sweatman)
Subject: Re: seq. gel problems
Organization: MRC Human Genome Resource Centre
Distribution: bionet
Date: Tue, 10 Nov 1992 14:04:28 GMT

In article <9211100553.AA18546@pelican.dbe.csiro.au>, adeno@dbe.csiro.au (both lab) writes:
> We are having problems with sequencing gels lately, we see a lot of smearing
> or patches where the ladder is fuzzy.  Any suuggestions ?
> Thanks for your advice.
> Adam


Make sure when you're pouring your gels that there is nothing vibrating the 
bench you're working on.
Someone in our lab had a similar problem, that only seemed to occur if he
poured the gel while someone else was using a microfuge.
I don't know what happens, perhaps it causes disruption to the
polymerisation of the gel.
From: adeno@dbe.csiro.au (both lab)
Newsgroups: bionet.molbio.methds-reagnts
Subject: seq. gel problems
Date: 10 Nov 92 21:53:28 GMT
Distribution: bionet

We are having problems with sequencing gels lately, we see a lot of smearing
or patches where the ladder is fuzzy.  Any suuggestions ?
Thanks for your advice.
Adam
From: agoodrid@vaxa.weeg.uiowa.edu (Stephen Klautky)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: sequencing from 300-500 bp
Date: 13 Nov 92 20:56:00 GMT
Organization: University of Iowa, Iowa City, IA, USA
News-Software: VAX/VMS VNEWS 1.41

In article: <1992Nov13.112454.942@unine.ch>
Jean.Mariaux@zool.unine.ch writes:

>In article <12NOV199221343137@vaxa.weeg.uiowa.edu>,
>agoodrid@vaxa.weeg.uiowa.edu (Stephen Klautky) writes:

>> 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. Everyone else had technical problems either pouring
>>>such a gel or taking it off the glass.

>>Sometimes it does seem to be harder to get the gel
>>off the plate onto the gel backing paper than the usual
>>A/B gel.  Generalization - It seems that if you have a little
>>trouble with sticky gels on plates with the regular mixes then
>>your troubles will increase; if you have no such problems then
>>Long Ranger should not give you problems.  This is just my
>>impression your observations may be different.

>>One annoying problem which has occured a few times - the gel
>>does not stick to the gel backing paper!  I had to put the
>>paper on the gel, invert, carefully lift the glass plate off
>>leaving the gel on the paper.


>Two shorts remarks:
>-I have noticed that it is really important to let the Long Ranger
>gel get really cool before trying to lift the plates. Just wait at 
>least 20 minutes (or enough time for the plates to reach room 
>temperature) and there should be no problem !
>-We also used to put some "Rain X" (a product intended to
>protect windshields from mist, easy to find in supermarkets) on one
>plate. That should prevent the gel sticking problems.
>Hope that helps!

Thanks for the information.

Next time I sequence I'll give it a longer resting time.  Usually
we let it sit for 10-15 min (protocol says 10).

As for sticky gels - we silanize the short plate with
dichlorodimethylsilane in CHCl3.  I haven't tried Rain-X.  Someone did
try Sigmacote and during some of her runs the gel would expand
(presumably absorbing liquid) messing up the experiment.  I have no
explanation for why this happened.

Steve     AGOODRID@VAXA.WEEG.UIOWA.EDU
From: rupp@embl-heidelberg.de
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Oligos for sequencing...
Date: 13 Nov 92 10:43:18 GMT
Organization: EMBL, European Molecular Biology Laboratory

In article <11NOV92.12163090@wums.wustl.edu>, wetsel_r@wums.wustl.edu writes:
> 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

ReHi Netters,
I'm involved here at EMBL in the DNA synthesis/sequencing service and in
my hands a simple n-Butanole precipitation does the job after the 
synthesis:

Dry oligo down after deprotection o/n in a speedvac, dissolve it in
90 ul of water, add 1000 ul n-Butanole, vortex vigorously and spin
it down in a table top centrifuge.
You'll get a nice pellet which has to be "speedvaced" again to 
remove all the n-ButOH (would inhibit the enzyme!).

The above mentioned procedure precipitates almost 100% of the oligo 
whereas by EtOH purification you might loose some of your product.
It was adapted from:

                   M.Sawadogo, M.W.VanDyke
                   NAR 1991 Vol19, No3, p 674

and slightly changed.

Fell free to contact me for further information,

Thomas

RUPP@EMBL-Heidelberg.de
From: Jean.Mariaux@zool.unine.ch
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: sequencing from 300-500 bp
Date: 13 Nov 92 11:24:53 GMT
Organization: University of Neuchatel, Switzerland

In article <12NOV199221343137@vaxa.weeg.uiowa.edu>, agoodrid@vaxa.weeg.uiowa.edu (Stephen Klautky) writes:
> 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
> 
> Some of the people in our lab use/have tried the Long Ranger
> gel mix.  Sometimes it does seem to be harder to get the gel
> off the plate onto the gel backing paper than the usual
> A/B gel.  Generalization - It seems that if you have a little
> trouble with sticky gels on plates with the regular mixes then
> your troubles will increase; if you have no such problems then
> Long Ranger should not give you problems.  This is just my
> impression your observations may be different.
> 
> One annoying problem which has occured a few times - the gel
> does not stick to the gel backing paper!  I had to put the
> paper on the gel, invert, carefully lift the glass plate off
> leaving the gel on the paper.
> 
> From what I can see the Long Ranger has two major advantages:
> First, it runs faster than the standard mixes.  Unfortunately,
> we did not see the advertised increase in resolution - but you
> know how that can vary - almost day to day.  Secondly, you don't
> fix the gel.  Of course, I know it has become fashionable not
> fix the ordinary gels (but in my experience this practice gives
> you a lower signal).
> 
> Some of my collegues had major problems with the LR gel sticking
> to the long plate and complain a bit.  I have good plates and the
> whole process goes easy for me (mostly) - except when people
> walk off with my plates.  :-)
> 
> Steve     AGOODRID@VAXA.WEEG.UIOWA.EDU
> 
Two shorts remarks:
-I have noticed that it is really important to let the Long Ranger 
gel get really cool before trying to lift the plates. Just wait at 
least 20 minutes (or enough time for the plates to reach room 
temperature) and there should be no problem !
-We also used to put some "Rain X" (a product intended to
protect windshields from mist, easy to find in supermarkets) on one 
plate. That should prevent the gel sticking problems. 
Hope that helps!

Jean       (jean.mariaux@zool.unine.ch)
Newsgroups: bionet.molbio.methds-reagnts
From: zxmkr08@studserv.zdv.uni-tuebingen.de (Cornelius Krasel)
Subject: Re: Aqua Bidest
Organization: Comp. Center (ZDV) U of Tuebingen, FRG
Date: Wed, 11 Nov 1992 19:07:52 GMT

Germans usually also refer to Millipore-purified water as "aqua bidest".
If you need extra-pure water: Merck sells some water which is specially
designed for HPLC.

--
/* Cornelius Krasel, Department of Physiological Chemistry, U Tuebingen    */ 
/* email: krasel@studserv.zdv.uni-tuebingen.de (Internet)   HAS CHANGED!!  */
/*        NO WIN/X400 MAIL POSSIBLE AT THE MOMENT!                         */
/* "People are DNA's way of making more DNA." (anonymous)                  */
Newsgroups: bionet.molbio.methds-reagnts
From: bchs1b@Elroy.UH.EDU (Michael Benedik)
Subject: Re: What causes the plaque size difference?
Reply-To: bchs1b@Elroy.UH.EDU
Organization: University of Houston
Distribution: bionet
Date: Thu, 12 Nov 1992 06:16:21 GMT

In article <921111090655.2e58@VTHVAX.TAMU.EDU>, MCAO@VTHVAX.TAMU.EDU writes:
>Hi, Dear netters: I raised a question two days ago about the "phage size". I am
>sorry I did not make it clear. Here phage size I mean plaque size. Each time
>when I grow the phage bearing the cDNA library, the size of the plaque varies.
>					Ming Cao

There are a number of parameters which affect plaque size. Different
phages in a library will certainly have different plaque sizes depending
upon the insert size and probably the base sequence to some extent.
Other parameters that affect plaque size for a given phage stock
include the genotype of the host strain, the growth conditions of the
host and age of the plating cells, the number of plating cells you
use, the wetness of the plates you actually use, the temperature and
time you adsorb phage to cells. Among other factors.

If you want big juicy plaques the approach is: 1) use fresh plating
cells grown with maltose, 2) be sure it is a healthy genotype for
phages like C600 or LE392 (many others also ok), 3) use FRESH poured
plates (2-3 days) which are still very moist, 4) minimize the number of
cells you use for the lawn, 5) use tryptone plates instead of LB

Not all of these are essential, but these are the general rules for
consistent large plaques. For small plaques do the opposite. The use
of more plating cells is the most reproducible way to decrease plaque
size, or the choice of a poor genotypic strain.


----------------------------------------------------------------------
 Michael Benedik				INTERNET: Benedik@uh.edu
 Dept. of Biochemical & Biophysical Sciences	
 University of Houston				BITNET: Benedik@uhou
-----------------------------------------------------------------------
From: mcp@canctr.mc.duke.edu (Laura J. Weislo)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: seq. gel problems
Date: 10 Nov 92 16:47:44 GMT
Reply-To: mcp@canctr.mc.duke.edu
Distribution: bionet
Organization: Duke U. Med. Ctr., Durham, NC

In article <9211100553.AA18546@pelican.dbe.csiro.au>, adeno@dbe.csiro.au (both lab) writes:
>We are having problems with sequencing gels lately, we see a lot of smearing
>or patches where the ladder is fuzzy.  Any suuggestions ?
>Thanks for your advice.

>Adam

	AH!  We had the exact same problem in my old lab and it took
eons and eons to figure out why, so you fortunate, resourceful folks
can benefit from our stupidity..... We were using Pledge to keep the
gel from sticking to the plates, and Pledge, unlike Endust, accumulates
that waxy buildup that must have screwed up the polymerization or something.
A thorough soaking in chromic acid took care of the problem.
GOOD LUCK!

************************************************************
	!!!!			Laura J. Weislo
	 ^^			Duke University
	 @@			Dept. of Chemistry
	  )			Weisl001@dukemc.bitnet
	 0			Weisl001@mc.duke.edu
	"YOU'RE MY FORTUNE COOKIE PRIZE" - BEAT HAPPENING
************************************************************
From: greend@har-rbu.mrc.ac.uk
Newsgroups: bionet.molbio.methds-reagnts
Subject: Gell densitometry
Date: 13 Nov 92 09:15:42 GMT
Distribution: bionet
Original-To: methods@uk.ac.daresbury
Original-Cc: oneilp@uk.ac.mrc.har-rbu

Can anyone recommend micro based equipment and software for
gell densitometry. Preferable ibm-pc based. Alternatively can
anyone point to a review.

Dave Green
MRC Radiobiology Unit
Chilton
Didcot
Oxon                                       greend@uk.ac.mrc.har-rbu
From: bchs1b@Elroy.UH.EDU (Michael Benedik)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Which salt of X-P to use for detection of alkaline phosphatase?
Date: 12 Nov 92 16:08:26 GMT
Reply-To: bchs1b@Elroy.UH.EDU
Distribution: bionet
Organization: University of Houston

In article <01GR2UC9PTO2934TI6@INVERMAY.CRI.NZ>, VANNESTEJ%RUA1@INVERMAY.CRI.NZ writes:
>
>
>	Hello,
>
>
>	Which salt of X-P to use for detection of alkaline phosphatase?
>
>	To know if the gene I cloned contains a signal peptides sequence, I mutagenized 
>the gene with a transposon that carries a phoA gene derivative that lacks signal 
>peptides sequence.  The next step is the detection (or the non detection) of the 
>alkaline phosphatase, this is easily done (according to the publications) by 
>incorporing in the medium the 5-bromo-4-chloro-3indolyl-phosphate,also called X-P 
>or X-phosphate.  In the catalogue of Boehringer Mannheim (the only distributor of 
>X-P I found) there is two salts of X-P that are listed; a disodium salt and a 
>4-toluidine salt. The local representatives could not tell me which salt I should 
>use.  Can anybody help me?  Could you please also let me know if you know of 
>another source of the compound than Boehringer Mannheim.
>
>	Thank you,
>
>	Joel
>
>
>------------------------------------------------------------------------------
>  Joel L. Vanneste
>  The Horticulture and Food Research Institute of New Zealand Ltd 
>  Ruakura Agricultural Centre,		
>  Private Bag 3123, Hamilton,		e mail: VANNESTEJ@RUAKURA.CRI.NZ  
>  New Zealand						fax 64 7 838 5073
>------------------------------------------------------------------------------
>
>

We use the toluidine salt in my lab which works just fine. I don't know
if it really matters or not, but this does work. Lots of places sell
it, including sigma, biorad and numerous others. Genetics folks call
it XP but immunology folks call it BCIP used for detection of
western blots etc with alkakline phosphatase conjugated antibodies. 
BCIP and XP are one and the same...


----------------------------------------------------------------------
 Michael Benedik				INTERNET: Benedik@uh.edu
 Dept. of Biochemical & Biophysical Sciences	
 University of Houston				BITNET: Benedik@uhou
-----------------------------------------------------------------------
From: phjf@ariel.ucs.unimelb.EDU.AU (Patrick Helmer James Falckh)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Ligation techniques
Date: 6 Nov 92 01:19:13 GMT
Organization: University of Melbourne
NNTP-Posting-Host: ariel.ucs.unimelb.edu.au



Hi Bio-netters,
	I am looking for references on how to perform ligation of cDNA and
single strand oligos.  I am not a biochemist but a pharmacologist muddling my 
way throught molecular biology techniques and utilise RT/PCR to 
investigate changes in the expression of receptor types; and subtypes.  I
have seen several different ways to quantitate PCR products and have designed
my own standard that I wish to construct.

	My approach is two fold : 
	1)  To join several oligos together with some "junk" in between, that
will be inserted in Bluescript with T3 and T7 promoters; the idea is similar
to that of Wang et al (Proc. Natl. Acad. Sci USA, Vol 86:9717-9721). This
will allow me to create cRNA that can be included in the initial RT reaction
and adjust for efficiency;
	2)  To 'cut and paste' several sections from different sequenced
segments of PCR products that I presume will require the use of several
different restriction enzymes and linkers/adapters.

	From my reading both methods seem feasible; if not practical.

	Anyone with information (or constructive criticism) on how to go
about the construction of either two methods, or where to get the info from
journals would be greatly appreciated.



++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++
+                               |                                +       
+    Patrick HJ Falckh PhD      |                                + 
+    Dept of Pharmacology       |   Telephone : 61-03-344 6992   +  
+    University of Melbourne    |    Fax No   : 61-03-347 1453   + 
+    Parkville, Victoria        |                                +  
+    AUSTRALIA  3052            |                                +  
+                               |                                +  
+----------------------------------------------------------------+ 
+            "No matter how tall your grandfather was            +  
+                 you have to do your own growing"               +  
+                                                                + +
++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++
From: bhjelle@carina.unm.edu
Newsgroups: bionet.molbio.methds-reagnts
Subject: PCR Mutagenesis
Date: 12 Nov 92 22:50:38 GMT
Organization: University of New Mexico, Albuquerque

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
From: mic296e@vaxc.cc.monash.edu.au
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re:Laura, Overexpression of Eukaryotic proteins
Date: 6 Nov 92 04:07:07 GMT
Organization: Computer Centre, Monash University, Australia

Dear Laura,
having myself problems with the pMal system, I would greatly appreciate
if you could give a brief summary of the respondes you got.
In particullar, what does "carbenicillin" do that ampicillin doesn't.
In addition to what you described I (my protein) have severe degradation
problems with both pMAl p2 and c2. Degradation is occuring 
already the cell and the "fusion protein" has just the size of the MBP part
(42 kD).
Where can I get the E.coli strain BL 21 from and are there other strains or 
tricks to get this system going?

thanks:  Beat von Kanel
From: gessler@convex.HRZ.Uni-Marburg.DE (Gessler Manfred Dr.)
Newsgroups: bionet.molbio.methds-reagnts
Subject: reference manager
Date: 12 Nov 92 07:59:42 GMT
Distribution: bionet
Original-To: methods@uk.ac.daresbury


Does anybody know about the advantages/disadvantages of the several reference
managing programs for DOS systems like Reference Manager, Ref-11, Payrus etc.
What are the disk space requirements for the growing databases? 
Any recommendations or comments are welcome!

Manfred Gessler

_____________________________________________________________________________
Manfred Gessler                             phone:   (49)-6421-283584
Inst. f. Humangenetik                       FAX:     (49)-6421-285630
Bahnhofstr. 7A                              e-mail:
D-355 Marburg / Germany                     gessler@convex.HRZ.Uni-Marburg.de
-----------------------------------------------------------------------------
From: mm6y+@andrew.cmu.edu (Morris F. Manolson)
Newsgroups: bionet.molbio.methds-reagnts
Subject: sequencing from 300-500 bp
Date: 11 Nov 92 20:26:02 GMT
Organization: Carnegie Mellon, Pittsburgh, PA

Dear Netters,  I am sure someone must have asked this before but......

I am trying to sequence off of one reaction 300 to 500 bps using the 
standard IBI or BRL 44 cm glates.  Without having to resort to ultra-long
plates (of which I don't have), does anyone have any suggesting on how to
improve the resolution of the bands in the 300 to 500 bp region.  My reactions
are fine and I can see bands up to 600 bp from the primer, I just can't
resolve them well enough to read.  I am using 6% acrylamide wedge gels and run
them for 24 hours at 900 Volts, this gives me nice sequence on average
up to 350 bp,  on a great gel I can get 400.  I remember some company trying
to sell an alternative matrix to acrylamide, (of course I don't remember
who or where) which they claimed improved resolution, does anyone
out there know what I am talking about?  Thanks in advance, Morrie
From: ah690549@mbcr.bcm.tmc.edu (Annette C. Hollmann)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: sequence-specific protease
Date: 13 Nov 92 04:32:19 GMT
Distribution: bionet
Organization: Baylor College of Medicine, Houston, Tx
NNTP-Posting-Host: mbcr.bcm.tmc.edu

Pierce has a good selection of sequencing grade proteases, and the prices are
reasonable. I got TPCK trypsin from them, but I am still in the debugging
stage so I don't know yet how well the stuff works. E-mail me in about 2
months for an update - I should have the ancient DeSaga TLE unit optimized
by then.

Annette
ah690549@mbcr.bcm.tmc.edu

P.S. If you buy an enzyme from Pierce and it doesn't work as
advertised,they refund your money. I have never had to take them up on it,
but it is good to know that option exists.
From: leonfdo@CCWF.CC.UTEXAS.EDU (@ccwf.cc.utexas.edu, Leon F. Garcia)
Newsgroups: bionet.molbio.methds-reagnts
Subject: MEDLINE
Date: 7 Nov 92 19:43:45 GMT
Distribution: bionet

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

thanks.

Leon F. Garcia
Univ. of Texas, Austin
leonfdo@ccwf.cc.utexas.edu