Methods Digest, Vol 42, Issue 13, alkaline lysis -- unrealistic spec readings

Virash Gupta via methods%40net.bio.net (by virashkgupta from gmail.com)
Fri Nov 21 00:11:22 EST 2008


Hi,
If there is  no genomic DNA or RNA contamination, the high spec reading may
be due to high protein contamination. Check for A260/A280 ratio. Minipreps
are known for high protein contamination- not due tp protocol but due to
error in handling. After adding neutralizing buffer- sod/ pot acetate, due
not vortex or shake high, just mix by inverting many times, keep in ice for
5 min and spin at 12-13 K for 10 min or in cold centrifuge for 5 min.- here
we need a firm pellet. While transferring supernatant, take care in
transferring any ppt (full of proteins and genomic DNA). Otherwise your
plasmid DNA yield is quite good- so reagents are OK. We all know that it is
proper handling of technique which matters in this science. Besides 2 ug/ul
DNA (spec reading) is 10 times the 200 ng/ul- it may be just a calculation
error- check it by applying appropriate dilution factor. Hope this will
solve. All the best.


On 11/20/08, methods-request from oat.bio.indiana.edu <
methods-request from oat.bio.indiana.edu> wrote:
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> Today's Topics:
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>   1. Re: running ssDNA (30-1000nt) in denatured conditions
>      (cutest_chemist from yahoo.com)
>   2. In-gel end repair and linker ligation (Wenyi Feng)
>   3. Affinity matrix for Arginine (Werner Straube)
>   4. alkaline lysis -- unrealistic spec readings (Ed Siefker)
>
>
> ----------------------------------------------------------------------
>
> Message: 1
> Date: Wed, 19 Nov 2008 00:10:16 -0800 (PST)
> From: "cutest_chemist from yahoo.com" <cutest_chemist from yahoo.com>
> Subject: Re: running ssDNA (30-1000nt) in denatured conditions
> To: methods from net.bio.net
> Message-ID:
>        <d4361072-4827-44b4-bd1b-0bc4d9cb7251 from a26g2000prf.googlegroups.com>
> Content-Type: text/plain; charset=ISO-8859-1
>
> On Nov 5, 12:51 am, TC <tracyscie... from gmail.com> wrote:
> > Hi all,
> >
> > I am trying to run DNA under denatured conditions. Has anyone run single
> > stranded DNA (30nt to 1000nt) under denatured conditions.
> >
> > I am trying to decide whether to run a low percentage urea acrylamide gel
> or
> > an alkaline agarose gel.  Any thoughts/ideas?  I am thinking maybe
> running a
> > low percentage (3-4%) urea acrylamide gel, but wonder that it may not be
> > able to resolve ssDNA above couple hundred nucleotides in length even in
> low
> > acrylamide such as 3-4%?  Any thoughts?  Has anyone run alkaline agaorse
> for
> > ssDNA 30nt-1000nt?  If so, may I ask for your conditions?
> >
> > THANKS!
> >
> > Tracy
>
> Hi...
> I m currently using 20% Polyacrylamide gel(PAGE)for ssDNA (20-30 nt)
> and 15% PAGE for ssDNA of 30-50mer.Its giving good results...Hope this
> will help u..For more lengthy strans i think u should try 8-10% PAGE.
>
> Regards
>
>
> ------------------------------
>
> Message: 2
> Date: Tue, 18 Nov 2008 22:50:17 -0800
> From: Wenyi Feng <wfeng from u.washington.edu>
> Subject: In-gel end repair and linker ligation
> To: methods from magpie.bio.indiana.edu
> Message-ID: <B8D3A42D-D76D-4BA6-AD9E-C9F593A3A455 from u.washington.edu>
> Content-Type: text/plain; charset=US-ASCII; format=flowed; delsp=yes
>
> Hello,
>
> Does anyone have experience with performing end-repair and adaptmer
> ligation of chromosomal DNA embedded in agarose plugs?  I would
> appreciate any suggestions for products as well as protocols.  Thank
> you in advance.
>
> Wenyi Feng
>
>
>
> ------------------------------
>
> Message: 3
> Date: Wed, 19 Nov 2008 19:52:03 +0100
> From: "Werner Straube" <straube from biochem.mpg.de>
> Subject: Affinity matrix for Arginine
> To: <methods from magpie.bio.indiana.edu>
> Message-ID:
>        <36B75E885C4376419EC0020DA151B41CE6CC00 from msx.w2k.biochem.mpg.de>
> Content-Type: text/plain;       charset="us-ascii"
>
> Hello everybody,
>
>
>
> I am looking for an affinity matrix that binds with high specificity to
> a C-terminal Arginine, but the binding should be also reversible.
>
>
>
> Any suggestions ?
>
>
>
> Thank you,
>
>
>
> Werner
>
>
>
> *********************************************
>
> Werner L. Straube
>
>
>
>
>
> ------------------------------
>
> Message: 4
> Date: Wed, 19 Nov 2008 15:54:10 -0600
> From: Ed Siefker <ebs15242 from creighton.edu>
> Subject: alkaline lysis -- unrealistic spec readings
> To: methods from magpie.bio.indiana.edu
> Message-ID: <49248B02.7020207 from creighton.edu>
> Content-Type: text/plain; charset=ISO-8859-1; format=flowed
>
> We ran out of miniprep columns, so I'm trying out the alkaline lysis
> protocol in Maniatis (CSH).  When checking the DNA on my spec, I get
> a nice shaped curve, with good ratios, but the concentration is
> unrealisticly high. I'm getting readings of around 2 ug/ul.  When I run
> it on a gel and actually visualize the DNA, it can't be more than
> 200ng/ul tops.
>
> I can't figure this out.  My spec still reads known DNA concentrations
> correctly. There aren't any smears or anything that would indicate genomic
> DNA or RNA contamination.  This DNA digests just fine with EcoRI too.
>
> Anyone have any idea what's going on here?  I need to have these plasmids
> sequenced, but I'm not sure what to tell them the concentration is.  Thanks
> -Ed
>
>
>
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> End of Methods Digest, Vol 42, Issue 13
> ***************************************
>



-- 
Dr V K Gupta
Sr Microbiologist (Molecular Biology)
Insect Molecular Biology Lab
Department of Entomology
Punjab Agricultural University
Ludhiana (Pb)-141004- India
M: 09815963210


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