HDTenEightyP/NetNews huggingface dataset:
From: huhta@poe.ccs.itd.umich.edu (Christopher T. Huhta)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Background: lambda gt11
Date: 26 Jan 93 18:55:25 GMT
Organization: University of Michigan - College of Literature, Science, and TheArts
NNTP-Posting-Host: poe.ccs.itd.umich.edu

I have tried doing plaque lifts from a gt11 cDNA library on
Nitorcellulose filters.  I have the problem of VERY high background after
I wash my membranes.  I block with ssDNA (Herring Sperm).  I believe that
I am washing the membranes incorrectly or something.  This is my first
attempt at screening such a library, and there is nobody around my lab who
has done it before to help me diagnose the problem.  

After 2-3 perhybridization at 42 degrees, P-32 labeled probe is added and
allowed to hybridize overnight at 42.

The next day I wash the membranes (low stringency: 2x SSC, 0.1% SDS).  I
wash in this fashion:  I open the heat sealed bag with my 'branes in it,
place them (approx. 10) in a 6x9 rubbermaid container with about 500 mL of
2x SSC, 0.1% SDS.  I separate the filters so they don't stick to one
another, and rock them.  I drain the wash fluid and pour another 500 mL
directly back into the dish for the next wash.  I do two 15 min washes at
RT, and two at 50 degrees.

Changing SDS and SSC conc. doesn't change anything.  I must be doing
something wrong.  My membranes (after autoradiography) come out with some
clear ares (shoing some hybridization where appropriate) and the rest of
the membrane is black as night.  

I might add that I am but a first year med student doing research, and
although I think I have a good grip on my laboratory skills, I don't do
this as a living, and thus my knowledge is somewhat limited.

Thanks in advance!

Chris Huhta
huhta@milton.ccs.itd.umich.edu
From: GIETZ@bldghsc.lan1.umanitoba.ca
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Insert Positive blue col
Date: 21 Jan 93 20:29:00 GMT
Distribution: bionet

We have also seen that in many clonings into the pUC 
lacZ MCS we end up with quite a few yeast DNA
fragments that give various shades of blue.  
If we get just blue  (no shade differential)
and white colonies the blues are usually non inserted.  But if we see 
one or two different shades of Blue clonies, including whites,
 on a plate it appears that the whites represent one orientation 
of the insert in the MCS and the lighter blue represents the 
other orientation of the fragment in the MCS.  On occasion I have
run across some fragments (1.1 kb URA3 Hind-Sma) that  are as blue 
the non-inserted vector in one orientation (blunt end ligation) and white
in the opposite ori.  One way we can sort of tell the difference between
inserted and non-inserted blues with  pUC plasmids grown in 
DH5 alpha is that the non-inserted blues do no grow very well giving 
very small colonies, were as the inserted blues tend
to be bigger healther looking colonies.  This works most of the time
but not always.  It's insert (DNA fragment) dependant.

Dan Gietz
U of Manitoba
GIETZ@BLDGHSC.LAN1.UMANITOBA.CA
From: BAKERK@FRIR.AFRC.AC.UK (Ken Baker)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: problems with mrna quantitation
Date: 26 Jan 93 10:39:00 GMT
Distribution: bionet
Original-To: METHODS@UK.AC.DARESBURY

>Fellow netters:

>I have recently analysed my results during a quantitative rna analysis.
>During my analysis I used rRNA as a standard for the year to compare my
>summer hormone mrna levels.  I am dealing with temperate fish so I was
>expecting the summer hormone rna's : rRNA levels to be high during periods of
>rapid growth and the opposite to be the case during the winter months.

>However, I found just the opposite to take place.  That is to say that the
>ratios were high during the winter and low during the summer.  This is sort
>of an indication to me that in, winter flounder, the fish which I am
>presently studying rRNA varies considerably over the year.  Would anyone
>know of another mRNA which remains constant throughout the year which I
>would be able to use as a standard.
>Also, I was wondering if anyone would know if it would be a good idea to
>add a foreign rna to the G.T.C. extraction mixture to show quantitative rna
>extraction when using different probes.

>thanx Juan

>E-mail  Jgill@kean.mun.ucs.ca

Some time ago, I was involved in a project to quantitate mRNA levels specific
for calbindin in chickens under various regimens of diet and hormone treatment.
We found that the best internal standard to use (in an mRNA dotblot) was oligo
dT to normalise for the total mRNA. One word of warning tho, we started off
(like many others) using an actin-specific probe, but found that actin levels
could vary widely with different treatments.


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)
*************************************************************************
Newsgroups: bionet.molbio.methds-reagnts
From: pnh@fcsparc6.ncifcrf.gov (Paul N Hengen)
Subject: Re: ABI sequencer: reaction clean-up ??
Summary: Mini spin-columns
Organization: Frederick Cancer Research and Development Center
Distribution: bionet
Date: Sat, 23 Jan 1993 01:19:26 GMT

To clean up the Dye-terminator reactions, we use a mini spin-
column exactly as you would for a radiolabelled DNA probe.
There are several versions that have been posted here in the
past for making your own. You should have no problem using them
for the ABI system.

This from Raj Shankarappa (bsh@med.pitt.edu):

================================================================================
We now routinely purify the excess dye from reactions using home (lab)-made
quick spin columns.  They may not overcome the tedium but they sure are 
cheap.  We make a hole in the bottom of 0.5 ml eppendorf using a 26 g needle
and add about 25 ul volume of zirconium glass beads.  Then I pour sephadex
G-50 in 0.3M NaAc (5g in 60 ml) to the top of the tube.  Stick the small
eppendorf inside a 2 ml tube and spin at about 500 rpm for couple of minutes.
Now the tube is transferred to a new 1.5 ml tube and the sample is added on
top.  Now I spin at about 1500 rpm for couple of minutes.  
The eluate is precipitated by atleast 3 volumes of isopropanol washed with
70% etoh and resuspended for loading.
================================================================================

Note: We simply dry the void in a roto-vac before loading.

Paul N. Hengen
National Cancer Institute
Frederick Cancer Research and Development Center
Frederick, Maryland 21702-1201 USA
From: William_D_WARREN@UMAIL.UMD.EDU (ww40)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Looking for non-coli vector
Date: 27 Jan 93 20:17:00 GMT
Distribution: bionet

G'day,

We are looking for a double stranded, closed circular DNA vector
that will not replicate in E coli.

What is needed is a system whereby we can do invitro manipulation and
easily reintroduce the DNA back into it's host which will then grow it
up with high fidelity so we can isolate large quantities (around 1-200ug)
of monomeric molecules. We are currently considering B subtilis plasmids
however their propensity for concatamerization, instability and poor
transformation are far from ideal for our purposes. What is important,
as mentioned above, is that they not replicate in E coli and are double
stranded closed circles.

Does anyone out there have any suggestions ???

BILL WARREN
Center Ag Biotech
University of Maryland
From: KNECHT@UCONNVM.bitnet (Dave Knecht)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: DNA purification
Date: 23 Jan 93 14:58:39 GMT
Distribution: bionet

In regard to JGraham's message about DNA disappearing after precipitation in th
ese purification schemes.  I have had the same experience.  Any ideas out there
 as to what is going on and how to prevent it? It is only a problem for me in t
rying to purify from agarose.  Is there a precipitatoin inhibitor in the agar?
DaveK
From: bchs1b@Elroy.UH.EDU (Michael Benedik)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Photography
Date: 28 Jan 93 18:38:03 GMT
Reply-To: bchs1b@Elroy.UH.EDU
Organization: University of Houston
NNTP-Posting-Host: elroy.uh.edu

In article <1993Jan28.142542.14090@newssrv.edvz.univie.ac.at>, A8101DAL@vm.univie.ac.at (Martin Hetzer) writes:
>Usually people use the POLAROID system to take pictures of their
>EB stained gels. Who has tried reflex camera for this purpose and
>which film type and sensitivity is recommended?
>Thanks in advance
>Martin

I know a number of labs who use either 35mm cameras or 3x5 sheet film instead
of polaroid. The labs I know opt for speed and use Kodak Tri-x film which
is more than sufficient for photographing gels. The main advantage is
cost and quality of photos which can be made for publication. The disadvantage
is that if you use 35mm roll film then you need to wait until the roll is
all shot in order to develop it (if you only take 1-2 pictures per roll
you lose the cost advantage). Sheet film is also pretty cheap and you
can develop each sheet as you make it.
Of course developing the film every time you take a gel is a big pain in
my opinion.
----------------------------------------------------------------------
 Michael Benedik				INTERNET: Benedik@uh.edu
 Dept. of Biochemical & Biophysical Sciences	
 University of Houston				BITNET: Benedik@uhou
-----------------------------------------------------------------------
From: jasons@bmc.uu.se (Jason E Stewart)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: insert-positive blue cols
Date: 21 Jan 93 20:22:55 GMT
Followup-To: bionet.molbio.methds-reagnts
Organization: Dept of Med Gen
NNTP-Posting-Host: mac4.medgen.uu.se

In article <1993Jan21.090729.79@ebc.ee>, mspeek@ebc.ee (Mart Speek) wrote:
> 
> 
> Hi Netters;
> 
> I would like to know whether anyone has encountered problems
> in traditional blue/white colony or plaque screening procedures
> (beta galactosidase assay) when sorting out (non)recombinant
> clones. My question is :-( Could relatively long inserts (>0.5kb)
> sometimes give rise blue (beta gal+) clones? How is it possible?
> Any theoretical or practical solutions (incl refs) to this 
> particular problem are welcome.
> 

I have had similar problems with cloning a 242 bp product (i.e. all
positive colonies were faint blue). I pressumed that it had to do with
in-frame insertion, so that the insert left the B-gal in frame while not
disturbing the proteins function. Since I was using a phosphatased vector
cut with two enzymes the background was almost non-existant, so I stopped
using blue-white screening. Perhaps the best recommendation I can make is
to use primers with different enzyme sites and to phosphatase your vector,
that way you won't need screening.

good luck,

jason

----------------------------------------------------------------
Jason Stewart    	                      Father at large
Department of Medical Genetics
jasons@bmc.uu.se
From: bsh@MED.PITT.EDU (Basavaraju Shankarappa)
Newsgroups: bionet.molbio.methds-reagnts
Subject: problems with deletions in HIV constructs.
Date: 27 Jan 93 20:23:42 GMT
Distribution: bionet

Dear fellow net-lookers
	I am having lot of problems with the deletions in the HIV constructs.
When I ligate pieces that correspond to pst-sal fragment, more than 50%
of my colonies have apparent deletions as seen by the presence of bands in
the 1.5 kb and 3.0 kb regions and nothing else, I mean the supercoiled forms.

I could minimize some of this problems after I switched to SURE strain from
Stratagene.  But now, even this has not been of any help.
I tried growing cells at 30 C and stop growth after
about 8 hours by adding chloramphenicol, but has not been of good help either.

	Can somebody working with HIV constructs, especially the pBRU3
help me here.  Some of the labs I checked with say they do not have this
problem.  I do not see any significant differences between my and their
procedures.  I basically follow the standard procedure using LB medium
with 100 ug/ml of ampicillin.  
	If there are any specific strains that work very well for this,
will it be possible for you to send it to me.
	Thanks in advance.
Raj Shankarappa
bsh@med.pitt.edu
Univ of Pittsburgh
Newsgroups: bionet.molbio.methds-reagnts
From: tjrad@iastate.edu (Thomas J Radosevich)
Subject: Re: insert-positive blue cols
Keywords: blue/white selection 
Organization: Iowa State University, Ames IA
Date: Thu, 21 Jan 1993 18:45:54 GMT

In <1993Jan21.090729.79@ebc.ee> mspeek@ebc.ee (Mart Speek) writes:


>Hi Netters;

>I would like to know whether anyone has encountered problems
>in traditional blue/white colony or plaque screening procedures
>(beta galactosidase assay) when sorting out (non)recombinant
>clones. My question is :-( Could relatively long inserts (>0.5kb)
>sometimes give rise blue (beta gal+) clones? How is it possible?
>Any theoretical or practical solutions (incl refs) to this 
>particular problem are welcome.

>M. Speek

>mspeek@ebc.ee (ESTONIA)

I've had blue positives twice--one of 143 bp and the other 550 bp.  For the 
143 I unsuccessfully checked white colonies for quite some time.  Eventually
I checked blue colonies and they were all positive.  Now, whenever I clone a
small fragment, I always throw a few blue colonies in the first batch of
minipreps.  (BTW, these were bluescript/XL1 blue transformations)

Hope it helps--Tom Radosevich
From: msw1633@sigma.tamu.edu (Mark S. Whitsitt, N5RJF)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Background: lambda gt11
Date: 27 Jan 93 16:36:00 GMT
Reply-To: MSW1633@SUMMA.TAMU.EDU
Organization: Texas A&M University, Biochemistry Department
NNTP-Posting-Host: sigma.tamu.edu
News-Software: VAX/VMS VNEWS 1.41

In article <1k41etINN4kl@controversy.math.lsa.umich.edu>, huhta@poe.ccs.itd.umich.edu (Christopher T. Huhta) writes...
>I have tried doing plaque lifts from a gt11 cDNA library on
>Nitorcellulose filters.  I have the problem of VERY high background after
>I wash my membranes.  I block with ssDNA (Herring Sperm).  I believe that
>I am washing the membranes incorrectly or something.  This is my first
>attempt at screening such a library, and there is nobody around my lab who
>has done it before to help me diagnose the problem.  
> 
>After 2-3 perhybridization at 42 degrees, P-32 labeled probe is added and
>allowed to hybridize overnight at 42.

I assume from the hybridization temperature that you are using a DNA probe.  Is 
that correct?  Also, what are you using for hybridization buffer?  I use the 
rapid hyb buffer from Amersham and it works great!  I have also used standard 
Denhardt's and other made from scratch buffers with similar results.  Also, 1 
hour of prehybridization at the hybridization temp should be more than 
sufficient no matter what buffer you use.  I sometimes do a 15 min pre-hyb with 
the buffer described in the Stratagene pBluescript exo/mung DNA sequencing 
system manual (ie, PE/formamide)

> 
>The next day I wash the membranes (low stringency: 2x SSC, 0.1% SDS).  I
>wash in this fashion:  ... 500 mL of 2x SSC, 0.1% SDS...  I do two 15 min 
>washes at RT, and two at 50 degrees.

I would recommend 2 15 min washes at RT with 2x SSC, and 0.5% SDS and then 2 
15-30 min washes at 50 degrees with 0.2x SSC/ 0.5% SDS.  This has always worked 
for me whether doing northerns or plaque lifts or such. The only changes are the 
temperatures for the last 2 washes.  Usually I wash at 60-65 degrees for a 
northern.  You'll have to play with it to get it just right.  If you increase 
the temp you will reduce the background, but you may make the wash too stringent 
if you are not careful.
> 
>Changing SDS and SSC conc. doesn't change anything.  I must be doing
>something wrong.  My membranes (after autoradiography) come out with some
>clear ares (shoing some hybridization where appropriate) and the rest of
>the membrane is black as night.  

>Chris Huhta
>huhta@milton.ccs.itd.umich.edu

Good luck...

Mark S. Whitsitt, N5RJF        Texas A&M University, Dept of Biochemistry
Internet:  msw1633@summa.tamu.edu 	  College Station, Tx. 77843-2128
AMPRnet:   n5rjf@n5rjf.ampr.org 			   (409) 845-0832
Packet/APLINK:  N5RJF @ KE5HE.TX.USA.NA
   "You can't throw darts when you're empty, man." - another Shadelism
From: wsun@jeeves.ucsd.edu (Fiberman)
Newsgroups: bionet.molbio.methds-reagnts
Subject: pGEX fusion vector expression
Date: 23 Jan 93 00:39:20 GMT
Organization: University of California, San Diego

I have just cloned my gene into the pGEX vector using XL1-blue
cells.  Now the question is this:  is XL1-blue a suitable cell
line for expressing my fusion protein?  If not, what is a good
cell line to use?  Anyone who have had experience with the pGEX
vector please respond.

-fm

ps.  pGEX is a glutathione S-transferase fusion vector.
From: Mark S. Rose <msr2@cornell.edu>
Newsgroups: bionet.molbio.methds-reagnts
Subject: Fungal Transformation (Read this one)
Date: 24 Jan 1993 20:17:59 GMT
Organization: Cornell University
Distribution: world
NNTP-Posting-Host: cu-dialup-0322.cit.cornell.edu
X-UserAgent: Nuntius v1.1.1d7
X-XXDate: Sun, 24 Jan 93 15:26:23 GMT

Has anyone attempted to transform either Mycosphaerella zeae-maydis
(anamorph: Phyllosticta maydis) or any of the non-Cochliobolus
Helminthosporium spp. ?

			                                                Thanks
                                                       Mark
msr2@cornell.edu
Dept. of Plant Pathology, Cornell U.
Ithaca, NY, USA
From: scolnipa@esvax.dnet.dupont.com
Newsgroups: bionet.molbio.methds-reagnts
Subject: (none)
Date: 25 Jan 93 14:02:18 GMT
Distribution: bionet

I'm running fairly long (52 cm) sequencing gels with labelled DNA
fragments.  Right now we have to cut the top 10 cm because we don't have
X-ray film that's long enough.  I recall seeing X-ray film in rolls
somewhere.  Does anybody recall brand, size and price?

Also, we don't have a gel dryer that accomodates long gels.  Any
alternative drying method?

Cheers everybody

Pablo A. Scolnik
scolnipa@esva.dnet.dupont.com
From: rbonfigl@waite.adelaide.edu.au (Rod Bonfiglioli)
Newsgroups: bionet.molbio.methds-reagnts
Subject: single stranded cDNA prep
Date: 25 Jan 1993 03:50:19 GMT
Organization: The University of Adelaide, Waite Campus
NNTP-Posting-Host: schooner.waite.adelaide.edu.au
Summary: method wanted for the generation of single stranded cDNA by pcr
Keywords: pcr sscDNA
Faculty: Agricultural & Natural Resource Sciences

does any one out there in netland know of a good method for generating single
stranded cDNA by PCR?  i have the methods for doing it by the m13 method, but i
would like to do it by pcr if poss.

thanks in advance for any suggestions

rod bonfiglioli
waite institute
--
Rod Bonfiglioli, Waite Agricultural Research Institute,
University of Adelaide, South Australia.
e-mail rbonfigl@waite.adelaide.edu.au
From: Robert.H.Gross@dartmouth.edu (Robert H. Gross)
Newsgroups: bionet.molbio.methds-reagnts
Subject: E. coli 16S rRNA clone
Date: 25 Jan 93 18:05:42 GMT
Organization: Dartmouth College, Hanover, NH
X-Posted-From: InterNews1.0b7@newshost.dartmouth.edu

Hi folks-

I posted a request similar to this one in December, but did not receive
any responses - probably because of the holiday season. I am interested
in obtaining a construct containing the E. coli 16S rRNA region,
preferably downstream from a T7 or SP6 promoter. Does anyone have such
a beast or know where I might obtain one? If not, is there available an
entire cloned ribosomal gene (16S + 5S + 23S) that I could use as a
starting point to make my own construct?

Thanks in advance for any help or leads.

Bob Gross
Dept. of Biology, Dartmouth College
bob.gross@dartmouth.edu
Newsgroups: bionet.molbio.methds-reagnts
From: jgraham@bronze.ucs.indiana.edu (the End)
Subject: Re: DNA purification
Organization: Indiana University
Distribution: bionet
Date: Sun, 24 Jan 1993 23:31:17 GMT

In <9301231503.AA04046@net.bio.net> KNECHT@UCONNVM.bitnet (Dave Knecht) writes:

>In regard to JGraham's message about DNA disappearing after precipitation in th
>ese purification schemes.  I have had the same experience.  Any ideas out there
> as to what is going on and how to prevent it? It is only a problem for me in t
>rying to purify from agarose.  Is there a precipitatoin inhibitor in the agar?
>DaveK




Yeah. What is that thing ?
I have however heard that big spenders have been able to concentrate the 
eluate with Amicon microfiltration columns. My original post also may have
suggested yields in the microgram/ microliter range. The abbreviation was
meant to suggest nanograms per microliter (10-50).

Jim
J. Graham
From: bchs1b@Elroy.UH.EDU (Michael Benedik)
Newsgroups: bionet.molbio.methds-reagnts
Subject: RE: Long Seq Gels
Date: 26 Jan 1993 05:42:47 GMT
Organization: University of Houston
Distribution: bionet
Reply-To: bchs1b@Elroy.UH.EDU
NNTP-Posting-Host: elroy.uh.edu

In article <2B643AFA@adminbldg.lan1.umanitoba.ca>, GIETZ@bldghsc.lan1.umanitoba.ca writes:
>------------------------------------------------------------------------------
>I'm running fairly long (52 cm) sequencing gels with labelled DNA
>fragments.  Right now we have to cut the top 10 cm because we don't have
>X-ray film that's long enough.  I recall seeing X-ray film in rolls
>somewhere.  Does anybody recall brand, size and price?
>
>Also, we don't have a gel dryer that accomodates long gels.  Any
>alternative drying method?
>
>Cheers everybody
>
>Pablo A. Scolnik
>scolnipa@esva.dnet.dupont.com
>_____________________________________________________
>
>Pablo, 
>    Why don't you try cutting the gel with a razor blade
>and putting your gel to film in pieces.  If you load a sample
>twice (at two different times) the place of the cut should
>be no problem ( Overlap with the other load).  This worked 
>for me when I was using King Kong gels.
>
>________________________________________
>Dan Gietz
>Department of Human Genetics
>University of Manitoba
>770 Bannatyne Ave, Rm 250
>Winnipeg, Man, Canada
>R3E 0W3
>GIETZ@BLDGHSC.LAN1.UMANITOBA.CA
>_________________________________________


I agree with Dan that cutting the gel is probably the simplest. But
long film does exist. A few years ago (I just counted and that means
8 years) we were running 1 meter sequencing gels and had obtained 1 m
long film and cassettes and even intensifying screens (the good old days).
The film was standard kodak x-omat. So I would directly contact kodak or
an appropriate kodak x-ray material distributor.


----------------------------------------------------------------------
 Michael Benedik				INTERNET: Benedik@uh.edu
 Dept. of Biochemical & Biophysical Sciences	
 University of Houston				BITNET: Benedik@uhou
-----------------------------------------------------------------------
From: Mark S. Rose <msr2@cornell.edu>
Newsgroups: bionet.molbio.methds-reagnts
Subject: Fungal Transformation
Date: 23 Jan 1993 16:59:44 GMT
Organization: Cornell University
Distribution: world
NNTP-Posting-Host: cu-dialup-0311.cit.cornell.edu
X-UserAgent: Nuntius v1.1.1d7
X-XXDate: Sat, 23 Jan 93 12:08:19 GMT


msr2@cornell.edu
Dept. of Plant Pathology, Cornell U.
Ithaca, NY, USA