HDTenEightyP/NetNews huggingface dataset:
From: steffen@mbcr.bcm.tmc.edu (David Steffen)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: labelling dog muscle cells (primary culture)
Date: 23 Dec 1992 18:49:15 GMT
Organization: Baylor College of Medicine, Houston, Tx
NNTP-Posting-Host: mbcr.bcm.tmc.edu

In article <168C69C29.BIOSEE@UKCC.UKY.EDU> BIOSEE@UKCC.UKY.EDU (Stephanie Edelmann) writes:
>As an infrequent reader of this group, I don't know if this question is
>appropriate in this context.

100% appropriate, IMHO (though see below*).

>Here's my problem.  I am supposed to label a primary dog muscle cell
>culture so that the cells can be recognized when they are reinjected
>in the same dog.
> 
>1. Is it feasable (sp?) to use a marker-plasmid to do that or
>are the cells going to be senescent by the time I have transfected
>and selected them?  (I have never worked with primary cell culture)

I have not heard of this being done successfully, but I *AM*
"speaking" off the cuff.

>2. Should I go to a retroviral vector?  They are supposed to be pretty
>selective in their host.  What vectors could be used in a dog?

This is what most people do.  I'm pretty sure that the standard
amphotropic vectors will work fine in dog cells.  Muscle cells might
present problems, however, as retroviruses can only infect replicating
cells.  Myoblasts might be ok, if that will do.

>3. Where could I find information of this kind?  Not only the above
>questions, but things like "general" transfection procedures etc.

We have looked for a good cookbook on these kinds of techniques and
have not found one.  This may be because the techniques change to
rapidly.  All I can suggest is a full blown Medline search.

Once you are searching, look for the new approaches which use
asialoglycoproteins and adenovirus to increase transfection
efficiency.  This might work well for muscle cells.

* You ask for email responses due to a lack of good access to bionet.
I sympathize, but strongly encourage you to try to improve this
situation.  In the first place, this topic is one for which an
_exchange_ on the newsgroup might be better than a simple collection
of replies.  Further, once you have collected email responses, it is a
nice thing to do to post a summary to the group for the help of
others.  Finally, you would be suprised at the number of good ideas
that come from following the group regularly.


-- 
David Steffen
Department of Cell Biology, Baylor College of Medicine, Houston TX 77030
Telephone = (713) 798-6655, FAX = (713) 790-0545
Internet = steffen@bcm.tmc.edu
From: muriana@aclcb.purdue.edu (Peter M. Muriana)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Plasmid DNA Extraction
Date: 30 Dec 92 16:30:33 GMT
Reply-To: muriana@aclcb.purdue.edu
Organization: Purdue University AIDS Center

In article <92364.221112U09723@uicvm.uic.edu>, Lixin Zhou <U09723@uicvm.uic.edu> writes:
>I got troubles in extracting plasmid DNA from Streptococcus
>pneumoniae (Gram-positive)......
>The troubles were: in both minipreparation (1-10 ml culture) and
>big-CsCl-preparation (3 Liters), I always could not separate
>plasmid DNA from 'chromosomal DNA' (or whatever else?).  In CsCl
>centrifugation tubes, I always got only one band which appeared not
                                    ^^^^^^^^^^^^^ 
>sharp (50Ti rotor, 48K rpm for 50-60 hrs, or even longer).  When I
>run DNA from that band on agarose gel, I saw both plasmid and
>'chromosomal DNA' (It appears to be chromosome or from chromosome.
>I will called it smear DNA)..........

>I repeated CsCl-preparation three times and miniprep several times.
>Every time, I could not separate plasmid DNA from cloudy DNA.
>
>Reagents including 2 N NaOH stock seem to work fine.  I successfully
>extracted 4.4-kb vector DNA (father of the 'un-extractable' plasmid)
>from a different strain of the same species by using the same reagents
        ^^^^^^^^^^^^^^^^^
    The "different strain" may not have the peculiarity of the strain in 
    question.  Don't Streptococcus pneumoniae make polysaccharide capsules?
    Perhaps this may be part of the problem? 	


>Could anybody see and tell me why I failed the separation?  What might
>the smear DNA be?  Thanks in advance.

>L Zhou
>Laboratory for Molecular Biology

	This may seem like a silly question to your question, but you are
	running Ethidium Bromide-CsCl gradients, not CsCl gradients, right?

	I experienced a problem with various Gram-positive genera in my 
	past where there was so much contaminating protein, etc., that
	went into the EtBr-CsCl gradient, that they reduced the level of
	EtBr available to intercalate into the pDNA/chrDNA, thereby not
	allowing effective separation of pDNA from chrDNA.

Peter Muriana
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Removing RNA from a mixture of protein and RNA
From: lihouba@dapsas1.weizmann.ac.il (Christo Houbaviy)
Date: Sat, 19 Dec 1992 20:27:04 GMT
Distribution: bionet
Organization: Weizmann Institute of Science, Computing Centre

In article <9212171610.AA02023@miranda.med.pitt.edu> bsh@MED.PITT.EDU (Basavaraju Shankarappa) writes:
>
>	Hi folks:
>
>	Sometime back there was a question about removing RNA contamination
>from a purified protein solution.  Among the many suggestions that were
>provided including precipitating proteins selectively some were neat.
>
>	There is one more way.  US biochemicals sells a large list of
>immobilized enzymes and RNase A is one of those.  I think this would be
>a good idea if you can purify the ribonucleotides away from the protein
>or if it does not matter.

I think that this is a _great_ idea. Getting rid of the
ribonucleotides should be no problem - one can use gel filtration or
ultrafiltration (e.g. Centricon).

>Raj Shankarappa
>Univ of Pittsburgh
>bsh@med.pitt.edu

Regards,

Christo

--
Christo Houbaviy [lihouba@dapsas.weizmann.ac.il]
Department of Molecular Genetics and Virology
The Weizmann Institute of Science, Rehovot, Israel
From: bl275@cleveland.Freenet.Edu (Dan Diaz)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: ? How to destroy DNA chemically (to reuse DNA columns)
Date: 24 Dec 1992 00:03:58 GMT
Organization: Case Western Reserve University, Cleveland, OH (USA)
Reply-To: bl275@cleveland.Freenet.Edu (Dan Diaz)
NNTP-Posting-Host: hela.ins.cwru.edu


In a previous article, djt2@po.CWRU.Edu (Dennis J. Templeton) says:
>
>You have a point, but our use here will consume ca $6000 per year.  Here's
>a comparison: would you throw away 10 FPLC columns a year at $600 each to
>sleep better at night?  maybe you or some would.  This is a variation of
>the "kit mentality" theme discussed here several times.  I think there are
>valid arguments on both sides of the discussion.

If you are spending these amounts of cash on columns then its time for you
to forget prepacked columns altogether and buy youself some resin, pack
your own columns, and sleep better at night.
-- 
Dizzy Dan         ddiaz@cwru.bitnet      bl275@cleveland.freenet.edu
Department of Molecular Genetics - Albert Einstein College of Medicine
"My opinions are my own, and they should be yours as well"
From: pamaga@biobase.aau.dk (Paulo Magalhaes)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Fast DNA from muscle biopsies
Date: 21 Dec 92 16:06:17 GMT
Organization: The Danish BioBase

Hi,

In the course of my work I have to extract DNA from muscle
biopsies. Since I have to deal with quite a number of samples,
I have set up a simple, fast and efficient method, basically
constructed from a number of published protocols (in short,
I homog. the biopsies, stew the homogenate in ProK, PhOH/CHCl3
extract, NH4Ac precipitate, EtOH wash and, presto, the DNA is
ready to be dissolved in T10E0.1!).

The weak point in the protocol (yeah, nothing is perfect...), is
yield. In general I have no problems, ending up with more DNA then
I need, even for complex analysis; sometimes, however, I have to
extract DNA from really _tiny, tiny_ (not to say ridiculously small!)
biopsies. It's then that trouble knocks on my door...

So, does anyone have a quick and efficient protocol for extracting
DNA from muscle biopsies?

Has anyone worked with the Qiagen columns? (using muscle tissue, of course!)

Thanks for all the info,

Paulo
RH
Copenhagen

PS - Should anyone want a more detailed version of the protocol I
now use, I'd be more then happy to share it - just drop me a line
to pamaga@biobase.aau.dk
From: hlhinkel@matt.ksu.ksu.edu (Heidi Lanora Hinkel)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Horseradish Peroxidase Blots
Date: 21 Dec 1992 08:29:06 -0600
Organization: Kansas State University
NNTP-Posting-Host: matt.ksu.ksu.edu

Does anyone out their store their horseradish peroxidase blots or blots
with horseradish peroxidase conjugated anitbody. If so what do you store
the blots in, under what conditions, and how long are the active.

					Thank you
					Heidi Hinkel
					hlhinkel@matt.ksu.ksu.edu
					Division of Biology
					Kansas State University
					Manhattan, KS 66506

"If you knew what you were doing you would probably be bored"
Organization: University of Illinois at Chicago
Date: Tuesday, 29 Dec 1992 22:11:12 CST
From: Lixin Zhou <U09723@uicvm.uic.edu>
Newsgroups: bionet.molbio.methds-reagnts
Subject: Plasmid DNA Extraction

I got troubles in extracting plasmid DNA from Streptococcus
pneumoniae (Gram-positive).  The plasmid is about 15 kb.
Pneumococcal chromosomal DNA and plasmid DNA are AT rich (about
62%).  The method I used is a modified alkali lysis protocol.
Solution II contains 1% SDS and 0.175 N NaOH instead of 0.2 N NaOH.
Solution III contains 50 mM Tris.Cl-pH8.0 in addition to 0.1 M
NaAc-pH4.8.

The troubles were: in both minipreparation (1-10 ml culture) and
big-CsCl-preparation (3 Liters), I always could not separate
plasmid DNA from 'chromosomal DNA' (or whatever else?).  In CsCl
centrifugation tubes, I always got only one band which appeared not
sharp (50Ti rotor, 48K rpm for 50-60 hrs, or even longer).  When I
run DNA from that band on agarose gel, I saw both plasmid and
'chromosomal DNA' (It appears to be chromosome or from chromosome.
I will called it smear DNA).  Even after I treated DNA from CsCl
prep with Solutions I, II and III for 2-3 times, I still could not
get rid of smear DNA.  Those results seem to be very reproducible.
I repeated CsCl-preparation three times and miniprep several times.
Every time, I could not separate plasmid DNA from cloudy DNA.

Reagents including 2 N NaOH stock seem to work fine.  I successfully
extracted 4.4-kb vector DNA (father of the 'un-extractable' plasmid)
from a different strain of the same species by using the same reagents
as used in extracting the 15-kb recombinant plasmid.  The recombinant
plasmid contains 10+-kb chromosomal DNA insert from a derivative strain
of the host.

Could anybody see and tell me why I failed the separation?  What might
the smear DNA be?  Thanks in advance.

L Zhou
Laboratory for Molecular Biology
Department of Biological Sciences (M/C 066)
University of Illinois at Chicago
From: cytogen@chmeds.ac.nz (Martin Kennedy)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: supercoiled DNA migration in agrose ?
Date: 27 Dec 92 20:37:05 +1200

In article <1992Dec23.111137.24858@wehi.edu.au>, chai_z@wehi.edu.au writes:
> 
> Hi,
> 
> 	Several batches of M13 RF DNA of mine have been cut by EcoRI or SmaI,
> etc. and then electrophoresized in agrose gel. Uncut DNA (supercoiled) of some
> batches migrated faster than the cut one (linear), while the others slower.
> Most likely, some batches of my DNA were nicked (open circular). Could any one
> tell me the correct agrose pattern of supercoiled, open circular and linear DNA?
> or tell me where to refer to?
> 
> Thanks in advance.
> 
> 
> Zhonglin Chai 


Some of your bands could be "miniphage" rather than various forms of cut/
uncut RF etc.  M13 throws off deleted derivatives (or miniphage) at a 
significant frequency.  They lack the multiple cloning and universal primer
site hence don't interfere with cloning or sequencing, but they can make it
hard to interpret M13 digestion patterns.

Cheers,

Martin
From: andrewh@uniwa.uwa.edu.au (Andrew Hobbs)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Acid washed sand
Date: 21 Dec 1992 13:54:06 +0800
Organization: The University of Western Australia
NNTP-Posting-Host: uniwa.uwa.edu.au
X-Newsreader: Tin 1.1 PL5

LAKSHMAN@MAINE.MAINE.EDU wrote:
: We are trying to isolate nuclei from a fungus (R. Solani)
: using a protocal which calls for "acid washed sand" to grind the
: mycelium. Does anyone know how to acid wash sand (we are trying 50%
: sulfuric acid overnight, is this appropriate)? Also why does it
: need to be acid washed? Perhaps to remove any metal ions?
: 
: Thanks,
: Dilip
: DILIP LAKSHMAN                             LAKSHMAN@MAINE.MAINE.EDU
: DEPT. PLANT BIOLOGY & PATHOLOGY
: UNIV. MAINE AT ORONO
: ORONO ME 04469

Hi, 

We routinely use acid washed sand for DNA extraction from plant
material.  We obtained the sand prewashed from BDH Chemicals and use
directly from the container.  If necessary we bake the sand in the oven
(ie to get rid of nucleases etc for RNA preparation).  The product
number from BDH is 33094.  I'm sure other manufacturers have similar
products. 

Andrew Hobbs
andrewh@uniwa.uwa.edu.au
From: DTANG@VM1.SI.USHERB.CA (Denis Tang)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Database search...
Date: 1 Jan 93 07:45:14 GMT
Distribution: bionet


                                                  Sherbrooke, 1er janvier 1993

      I am looking for places where I can do FASTA or BLAST searches for
proteins (is there another place than FASTA@EMBL-HEIDELBERG?).  Also, how can
I get the PIR sequence numbered P1;S20314 (I tried at NETSERV@EMBL-HEIDELBERG.DE
and FILESERVE@GUNBRF without luck)?  Is there something to replace the old
Genbank services like Fasta or Blast?

      Thanks in advance!

      Denis Tang (Dtang@vm1.si.usherb.ca)
      Universite de Sherbrooke
Newsgroups: bionet.molbio.methds-reagnts
From: maize@bragg.bio.purdue.edu
Subject: PCR using d(T) 19mer
Reply-To: maize@bragg.bio.purdue.edu
Organization: Purdue University, Dept. of Biological Sciences
Date: Sat, 2 Jan 1993 20:15:03 GMT

  I'm trying to do anchored PCR using only a dT 19mer and nested specific 
primers.  But (probably not surprisingly) dT19 is a cruddy PCR primer.  At 
42 oC annealing temp, I get no amplification, at 37 oC I get a smear at 
high molecular weights (like, above 1.6 kb.) and subsequent reamplification
with the nested primer gives me the same thing.  (The real band should be 
less than 600 bases.)  MgCl2 concentrations between zero and 9 mM have been 
tried.
  I'm trying to calibrate the rxns by using a previously cloned cDNA.  I 
use 5 pg in a rxn.  Amplification using either of the specific nested 
primers and a sequencing primer (the universal primer from the vector) gives 
me a clean band at the right size -- the oligo dT 19mer gives me the 
results I mention above.
  Any suggestions?  All accounts of anchored PCR I'm aware of either avoid 
using an oligo dT mer or don't mention any difficulty in using it (other 
than suggesting the use of nested internal primers.)
  Please email me, if possible, I'm out of town for a week and may not be 
able to read a posted response before it expires.

--Phillip
_______________________________________________________________________
Phillip SanMiguel               Purdue
maize@bilbo.bio.purdue.edu       Institute for
                                  Molecular    "We'll do anything
  Bennetzen Lab                    Plant          for funding."
                                    Science
_______________________________________________________________________
Newsgroups: bionet.molbio.methds-reagnts
From: plc@med.unc.edu (Philip L. Carl)
Subject: Sequencing Primers for Automated Sequencing
Originator: plc@cahaba
Organization: UNC-CH School of Medicine
Date: Tue, 22 Dec 1992 22:06:23 GMT

From: plc@med.unc.edu (Philip L. Carl)
Newsgroups: bionet.molbio.methds-regnts
Subject: Sequencing Primers for Dye Terminators
Followup-To: 
Distribution: world
Organization: UNC-CH School of Medicine
Keywords: 
I am posting this on behalf of a colleague who runs the automated
sequencing facility here.  She would like to obtain the sequence of
primers that have worked for priming vectors other than the M13 forward
and reverse primers.  She uses the ABI Taq sequencing system and would
particularly like to know about experiences with primers such as T3, T7,
SK, KS (for Bluscript vectors)  and SP6, and the lambda gt11 forward and 
reverse primers.  Please include any information about sequencing
conditions if they differ from the standard ABI protocols.  I believe
there is a new newgroup for automated sequencing questions, but I've lost
the address, so if someone could supply it and/or forward this notice to
that newsgroup I would appreciate it.  Thanks

Phil Carl
Program in Molecular Biology
and Biotechnology, UNC
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: None (HPRT Northerns)
From: cytogen@chmeds.ac.nz (Martin Kennedy)
Date: 22 Dec 92 13:34:33 +1200
Distribution: bionet

In article <9212210803.AA19833@net.bio.net>, URA1335@FRCITI51.BITNET writes:
> Hi! netters,
> 
> does anybody know the hybridation pattern of
> human fibroblast's HPRT mRNA on northern blots?
> normally mRNA=1,6 kb?
> 
>  I detect a band around 28 s RNA
> with a human HPRT cDNA
> 
> thanks for answers
> 
> Dr. Serdar ARAL
> E-Mail: "URA1335@FRCITI51.bitnet"


If you are using total RNA rather than poly-A RNA, maybe you're seeing cross
hybridization with the 28s - the vast excess of RNA in this region of the gel
sometimes gives a non-specific background signal.

Cheers,
Martin
From: djt2@po.CWRU.Edu (Dennis J. Templeton)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: supercoiled DNA migration in agrose [TBE vs. TAE or TPE gels]
Date: 28 Dec 1992 16:00:38 GMT
Organization: Case Western Reserve University, Cleveland, OH (USA)
Reply-To: djt2@po.CWRU.Edu (Dennis J. Templeton)
NNTP-Posting-Host: thor.ins.cwru.edu


In a previous article, vjongene@isrec-sun1.unil.ch (Victor Jongeneel) says:

>chai_z@wehi.edu.au wrote:
>
>: Hi,
>
>: 	Several batches of M13 RF DNA of mine have been cut by EcoRI or SmaI,
>: etc. and then electrophoresized in agrose gel. Uncut DNA (supercoiled) of some
>: batches migrated faster than the cut one (linear), while the others slower.
>: Most likely, some batches of my DNA were nicked (open circular). Could any one
>: tell me the correct agrose pattern of supercoiled, open circular and linear DNA?
>: or tell me where to refer to?
>
>: Thanks in advance.
>
>: Zhonglin Chai 
>
>
>In most agarose gels (but _not_ always), supercoiled circular DNA
>migrates fastest, followed by linear and nicked circles.  Linear does
>not always separate from supercoiled, nicked circles usually do.  This
>does of course not exclude the presence of miniphage (see other
>response in thread).  The best way to find out is to start
>with freshly purified (CsCl/EtBr) supercoiled DNA and treat it so as
>to make linears and nicked circles.
>
>Cheers,
>

To clarify Victors remark, the buffer used in Agarose gels affects the 
migration of superhelical plasmid DNA, and the other forms to a lesser 
extent. 

In TBE gels, form II (open circles) migrate slowest, and form I (CCC or
superhelical or supercoiled) migrates slightly faster. Form III migrates
fastest of all in TBE (that's full length linear DNA)

In tris acetate, or in the Tris phosphate buffer we prefer, form II
migrates slowest, with form III running just slightly ahead, and form I
(CCC) running far ahead of that.  The exact migration varies depending on
the size of the plasmid and the agarose concentration; in our 0.7% gels in
TPE, a 3 kb plasmid (supercoil, CCC) migrates near the 1.6 kb band from the
1 kb ladder.

We have never compared the migration in TBE and TPE directly, as it's hard
to run adjacent lanes of a gel in different buffers!

An added note, I would recommend saving your TBE for the acrylamide gels;
TPE can be made up at 50x and allows your gel pieces to dissolve in NaI for
the glass powder elution protocol.

good luck

dennis
From: Robert.H.Gross@dartmouth.edu (Robert H. Gross)
Newsgroups: bionet.molbio.methds-reagnts
Subject: 16S E. coli rRNA expression construct?
Date: 22 Dec 92 17:58:17 GMT
Organization: Dartmouth College, Hanover, NH
X-Posted-From: InterNews1.0b3@newshost.dartmouth.edu

Hi folks,

I am interested in obtaining a construct that has an in vitro
utilizable promoter (e.g. T7 or SP6) upstream of an E. coli 16S rRNA
"gene". The idea is to transcribe copies of the RNA in vitro. Does
anyone have such a beast they would be willing to share or know of a
place I might obtain it? If not in an expression vector, how about a
construct with the 16S region that I could transfer into an expressin
vector?

Thanks in advance and have a happy holiday season.

Bob Gross
Dept. of Biology, Dartmouth College
bob.gross@dartmouth.edu
From: URA1335@FRCITI51.BITNET
Newsgroups: bionet.molbio.methds-reagnts
Subject: (none)
Date: 21 Dec 92 08:03:04 GMT
Distribution: bionet

Hi! netters,

does anybody know the hybridation pattern of
human fibroblast's HPRT mRNA on northern blots?
normally mRNA=1,6 kb?

 I detect a band around 28 s RNA
with a human HPRT cDNA

thanks for answers

Dr. Serdar ARAL
E-Mail: "URA1335@FRCITI51.bitnet"
From: pamaga@biobase.aau.dk (Paulo Magalhaes)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Acid washed sand
Date: 21 Dec 92 15:51:39 GMT
Organization: The Danish BioBase

Hi, concerning

<LAKSHMAN@MAINE.MAINE.EDU> writes:

>We are trying to isolate nuclei from a fungus (R. Solani)
>using a protocal which calls for "acid washed sand" to grind the
>mycelium. Does anyone know how to acid wash sand (we are trying 50%
>sulfuric acid overnight, is this appropriate)? Also why does it
>need to be acid washed? Perhaps to remove any metal ions?

>Thanks,
>Dilip
>DILIP LAKSHMAN                             LAKSHMAN@MAINE.MAINE.EDU
>DEPT. PLANT BIOLOGY & PATHOLOGY
>UNIV. MAINE AT ORONO
>ORONO ME 04469

In my "previous life" I worked with _Neurospora crassa_ and one of the
things essencial in preparing DNA (for example) is grinding with acid
washed sand. I don't have the exact protocol in my head (we only did
it about once a year, with enough sand to last for quite a while...),
but if you're really stuck, I can dig it up for you. This posting,
however, was more of a note on a different key: you don't mention
the SIZE (average diametre) of the sand you use... In my experience
this is an important parameter, so do check it!

Anything you may need, just shout.

Regards,

Paulo
RH
Copenhagen
From: djs1539@cc.utah.edu (SEGAL, DAVID JAY)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Seeking psoralen tethered oligos
Date: 24 Dec 92 22:52:00 GMT
Organization: University of Utah Computer Center
News-Software: VAX/VMS VNEWS 1.4-b1

I am seeking a phosphoramidite derivative of psoralen which can be coupled 
to an oligonucleotide directly on the synthesizer column.  Apparently
there is such a derivative available from a company called Glen Research,
but this derivative only has a two carbon tether.  I need a 6 carbon
tether.  Ideally I would also like the linkage to be to carbon 5 or 8 of
the psoralen, rather than a furan carbon.

If anybody has any information on such a beast, or even knows what the
he*l I'm talking about, please E-MAIL me a reply.  Any info on a
psoralen I can tether to an oligo would be appreciated, journal refferences,
labs, companies... whatever.

Thanks, 
and have a Happy Holiday Season!!!!!:)

- Dave Segal
- Biochemistry, U. of Utah
- (801) 581-3848
- CarrollLab@medschool.med.utah.edu or
  dave.segal@m.cc.utah.edu