Methods Digest, Vol 37, Issue 14

Virash Gupta via methods%40net.bio.net (by virashkgupta from gmail.com)
Sat Jun 21 09:30:00 EST 2008


E.coli DH5-alha and sure are the best. High transformation efficiency
is practically obtained when after developing competency cells are
kept cold for atleast 24 hrs, and the stored cells are not older than
3 months. Plasmid copy number is decided both by the type of plasmid
vector and the size of insert DNA ( insert genes with proteinase
activity generally inhibit growth of transformed cells and need to be
grown in th epresence of a suitable proteinase inhibitor. The minimum
period for growth of E.coli clone from a single cell is 16 hrs-
generally if medium pH is near 7 a conspicuous clone of good size is
obtained in 20 hrs at 37C, although it  will be visible within 14-16
hrs. 4PM- 9AM period is not as suitable practically as if you get good
sized clone at around 9 or 10 AM, you will not have enough time on
same day to get good groth in LB for plasmid isolation. So let it grow
till 3 PM, inoculate in 3ml LB let grow overnight before you isolate
MP plasmid next day in the morning and analyze the same till evening.
For good yield of plasmid- while using alkaline lysis method- never
use old P1, P2, P3 solutions that are stored at RT for more than one
month. It is more true for Lysis solution which due to presence of
NAOH absorbs CO2 from atmosphere that results in decreased alkaline pH
to reduce lysis activity of this solution. Make solutions in bulk,
store the stock solutions at low temperature in tightened reagent
bottles- draw small working usable volumes in separate tubes for
regular use. It will solve many problems.
Low plasmid yields also result from improper handling of procedure
which results in residual endonuclease activities due to contaminating
(precipitated) proteins.

On 6/18/08, methods-request from oat.bio.indiana.edu
<methods-request from oat.bio.indiana.edu> wrote:
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> Today's Topics:
>
>   1. Re: Poor resolution when isolating ssDNA? (cheetah.zao from gmail.com)
>   2. Advice needed for Western blotting (Peter Ellis)
>   3. T4 PNK (peter)
>   4. Re: request for papers (Dr Engelbert Buxbaum)
>   5. Re: Advice needed for Western blotting (Dr Engelbert Buxbaum)
>   6. E.coli strain recommendation, please (DK)
>   7. doubt regarding agrobacterium DNA restriction digestion
>      (priyadarsini Tripuraneni)
>   8. Re: E.coli strain recommendation, please (Nick Theodorakis)
>
>
> ----------------------------------------------------------------------
>
> Message: 1
> Date: Mon, 16 Jun 2008 23:12:20 -0700 (PDT)
> From: "cheetah.zao from gmail.com" <cheetah.zao from gmail.com>
> Subject: Re: Poor resolution when isolating ssDNA?
> To: methods from net.bio.net
> Message-ID:
>        <5145efed-bf2d-45bd-be7d-f91b3dbca297 from w1g2000prd.googlegroups.com>
> Content-Type: text/plain; charset=ISO-8859-1
>
> 70% enthenol precipitation in the presence of 50 mM MgCl2 may work.
>
>
>
> On Jun 13, 5:56 am, Evan Reed <evan.g.r... from gmail.com> wrote:
> >         Hej,
> >
> >                 I am trying to isolate ssDNA after binding dsDNA oligos with
> > biotinylated antisense strands to strep microbeads, denaturing with
> > NaOH, then adding the supernatant containing the sense ssDNA to a
> > NAP-5 sephadex desalting column.  Unfortunately, I am getting terrible
> > resolution when I try to elute on the column -- even after 2 mL of
> > elution, I am still getting detectable levels of ssDNA in the eluate
> > (dsDNA oligos of the same length elute just fine in 0.5 mL, with
> > acceptable resolution).  The amount of DNA loaded into the column
> > should be well below the binding capacity so I am not sure why this is
> > happening.  Has anyone done this before?  Is there a better way to
> > purify the ssDNA instead of this column?
> >
> > Thanks,
> >
> > ER
>
>
>
> ------------------------------
>
> Message: 2
> Date: Tue, 17 Jun 2008 12:06:12 +0100
> From: Peter Ellis <pjie2 from cam.ac.uk>
> Subject: Advice needed for Western blotting
> To: methods from net.bio.net
> Message-ID: <Pine.LNX.4.64.0806171113570.2041 from hermes-1.csi.cam.ac.uk>
> Content-Type: TEXT/PLAIN; charset=US-ASCII; format=flowed
>
> Hiya,
>
> I am just starting up a project investigating two novel proteins in
> testis function.  My background is in molecular genetics and transcription
> profiling, so protein work is all pretty new to me.
>
> We have peptide antibodies prepared (or in the works) for both proteins -
> the first step is obviously to characterise the antibodies.  We're using
> peptide antibodies rather than antibodies to recombinant protein because
> there are many related genes, and we want to avoid cross-reactivity.
>
> We have ORFs cloned for each of the genes and their close relatives.  The
> aim is to use in vitro transcription/translation to generate pure
> proteins in order to check the antibody specificity.  Once we know
> that the antibodies do actually detect our proteins of interest (and not
> the relatives), we can move on to other more interesting questions!
>
> So the first plan is Western blots using whole testis extracts to see
> whether we have a band of the expected size in testis.  We'll then do
> further Western blots of testis extract alongside the various in vitro
> translated proteins in order to check AB specificity.  At this point I'm
> looking for advice / sanity checking on how to extract my proteins and
> what sort of gels to use for the Western blots.
>
> On looking around in the literature, I'm finding myself a little
> bewildered by buffer compositions - many papers refer to "standard RIPA
> buffer" or "standard Laemmli sample buffer", and yet there seem to be as
> many different recipes as there are laboratories.  Secondly, it looks like
> the most common buffers simply won't work for my proteins!
>
> Details below for each of the proteins of interest:
>
> *************************************************
>
> Protein 1: H2AL1
> ----------------
>
> This one is a novel histone.  I've looked at the QIAGEN QProteome Nuclear
> kit, but I'm loath to commit to using that as it doesn't tell you what any
> of the buffers are.  I can't even tell if it's an acid extraction or a
> salt extraction!  Also, it's very expensive for the amount of tissue you
> can process with it.
>
> So I'd prefer to do my own histone extraction if possible.  I believe that
> means I want to prepare purified nuclei and then do an acid extraction.
> Previously I've done nuclear preps when preparing high molecular weight
> gDNA - will the same buffers be appropriate for protein work?  Protocol is
> as follows:
>
> * Homogenise tissue at low speed in lysis buffer [0.05M TrisCl pH 7.5, 1mM
> EDTA, 5mM MgCl2, 50mM NaCl, 5% glycerol, 1% Triton X-100, 1% B-ME], using
> 100 mg tissue / ml buffer.
>
> * Centrifuge to spin down nuclei
>
> * Wash in lysis buffer and spin down again
>
>
> Once I've got the purified nuclei, the histone prep looks admirably
> straightforward.  According to Abcam, you just resuspend the nuclear
> pellet in 0.2M HCl and leave it overnight at 4 degrees C.  Centrifuge to
> spin down debris and keep the supernatant.  However, other protocols seem
> to use different acid concentrations, or H2SO4 instead of HCl, and may
> even include B-ME in the histone extraction buffer.
>
> One protocol I Googled called for you to neutralise the preparation with
> KOH afterwards - is that really called for?  Wouldn't it just drop the
> proteins straight back out of solution again?
>
> Once I've got my histone prep, what buffer should I use for running them
> on an SDS-PAGE gel?  Standard Laemmli sample buffer?  If so, *which*
> standard Laemmli buffer?  Will a normal SDS-PAGE gel work OK, or will
> there be pH issues due to the sample being in pretty much neat acid?
> Generally the lab uses Novex gels in a XCell mini-blot module.  For a
> histone (size ~17 kDa), it'll be quite a high percentage gel.
>
>
> *************************************************
>
>
> Protein 1:   mgclh
> ------------------
>
> Localisation of this protein is unknown, but a closely related gene
> (Gmcl1) is known to be a nuclear lamina protein.  Gmcl1 is RIPA-insoluble,
> so it's likely mgclh will also be RIPA-insoluble.  Protein size is
> expected to be around 55 kDa.
>
> The plan here is to do a standard protein extraction by homogenising
> testis tissue in RIPA + protease inhibitors.  Retain the supernatant (to
> get the RIPA-soluble fraction), and then dissolve the pellet in something
> (to get the RIPA-insoluble fraction).  Question is what to dissolve the
> pellet in.
>
> The original literature on Gmcl1 said they resuspended the RIPA-insoluble
> pellet in "SDS-polyacrylamide gel electrophoresis sample buffer", but
> doesn't give the composition.  I would assume this is 1x Laemmli sample
> buffer.  Does this sound plausible?  If so, what recipe for Laemmli buffer
> would you recommend?  I've found half a dozen different recipes, and the
> only common factor between them is ~60-100mM Tris pH 6.8 and 2% SDS.
> There are variants with and without DTT, with and without B-ME, with
> and without loading dye, using sucrose or glycerol for increased density!
>
> I would imagine I'll need to add B-ME to reduce the proteins before
> running on the gel, but should I add the B-ME at the point I resuspend the
> pellet, or only when I'm about to run an aliquot on a gel?  Will I need to
> add protease inhibitors, or will the Laemmli buffer itself take care of
> that?
>
> *************************************************
>
> Thank you for your time and patience - sorry for asking so many questions,
> most of which will undoubtedly have obvious answers.  Probably several
> mutually incompatible obvious answers, mind you, but that's biology for
> you.  My theory is that it's better to ask silly questions and look daft
> than to forge ahead without asking and cock something up!
>
> Thanks,
> Peter Ellis
>
>
> ------------------------------
>
> Message: 3
> Date: Tue, 17 Jun 2008 11:19:15 -0700 (PDT)
> From: peter <peter.ianakiev from gmail.com>
> Subject: T4 PNK
> To: methods from net.bio.net
> Message-ID:
>        <28b39b3f-6f52-4c5e-baa3-37a22181d61c from 8g2000hse.googlegroups.com>
> Content-Type: text/plain; charset=ISO-8859-1
>
> Hi all,
>
> have kinda silly question: Can T4PNK use dATP instead of rATP to
> phosphorilate DNA?
> Thanks ,
> Peter
>
>
> ------------------------------
>
> Message: 4
> Date: Tue, 17 Jun 2008 15:30:32 -0400
> From: "Dr Engelbert Buxbaum" <engelbert_buxbaum from hotmail.com>
> Subject: Re: request for papers
> To: methods from net.bio.net
> Message-ID: <op.ucwq46c866vu6s from bengelbert-dm.rusm.rossu.loc>
> Content-Type: text/plain; format=flowed; delsp=yes;
>        charset=iso-8859-15
>
> Am 12.06.2008, 04:53 Uhr, schrieb sheelu <gauravgene from gmail.com>:
>
> > Dear all friends:
> >        I have found two papers published in Nature & Science, the name
> > of which
> > are "How biotech can transform biofuels".(Nature biotechnology (2005
> > feb ) 26(2), 169-172; and another one is
> > "Fuel ethanol from cellulosic biomass" (Science 1991Mar 15:251(4999):
> > 1318-1323, I think
> > this paper would be great help to me.So anyone of here can help me to
> > get it?
>
> One method is to go to your library. If they do not have it, they can
> order a copy for you by interlibrary loan (not cheap though).
>
> The second method is to contact the authors and ask for a reprint. Their
> affiliation you get from the usual search tools, e.g. scholars.google.com
> or PubMed. Use email and you usually get an electronic copy within a few
> days. Exchange of reprints is a time-honored way of scholars to keep in
> contact.
>
>
> ------------------------------
>
> Message: 5
> Date: Tue, 17 Jun 2008 17:41:45 -0400
> From: "Dr Engelbert Buxbaum" <engelbert_buxbaum from hotmail.com>
> Subject: Re: Advice needed for Western blotting
> To: methods from net.bio.net
> Message-ID: <op.ucww7vua66vu6s from bengelbert-dm.rusm.rossu.loc>
> Content-Type: text/plain; format=flowed; delsp=yes;
>        charset=iso-8859-15
>
> Am 17.06.2008, 07:06 Uhr, schrieb Peter Ellis <pjie2 from cam.ac.uk>:
>
> > We have peptide antibodies prepared (or in the works) for both proteins
> > - the first step is obviously to characterise the antibodies.  We're
> > using peptide antibodies rather than antibodies to recombinant protein
> > because there are many related genes, and we want to avoid
> > cross-reactivity.
>
> That is fine, just remember that the epitope may be hidden in native
> proteins, thus the AB may not work e.g. in immunoprecipitation. In Western
> you deal with denatured proteins, so that is not a problem.
>
> > We have ORFs cloned for each of the genes and their close relatives.
> > The aim is to use in vitro transcription/translation to generate pure
> > proteins in order to check the antibody specificity.  Once we know that
> > the antibodies do actually detect our proteins of interest (and not the
> > relatives), we can move on to other more interesting questions!
>
> In addition to testing against individual proteins you should test against
> a crude tissue lysate. You should see only a single band. Even more
> telling would be a blot from a 2D-gel (SDS-PAGE after IEF).
>
> >  At this point I'm looking for advice / sanity checking on how to
> > extract my proteins and what sort of gels to use for the Western blots.
>
> The most common method is SDS-PAGE according to Laemmli (Laemmli: Nature
> 227 (1970) 680 and Laemmli & Favre, J. Mol. Biol. 80 (1973) 575-599).
> Separation is by molecular mass. For more specific detection, this is
> preceeded by isoelectric focussing, a method that separates by isoelectric
> point. See J. Klose and U. Kobalz: Electrophoresis 16 (1995) 1034-1059
>
> > On looking around in the literature, I'm finding myself a little
> > bewildered by buffer compositions - many papers refer to "standard RIPA
> > buffer" or "standard Laemmli sample buffer", and yet there seem to be as
> > many different recipes as there are laboratories.  Secondly, it looks
> > like the most common buffers simply won't work for my proteins!
>
> It's not as bad as that. The Laemmli buffer is a fairly good bet.
>
> > This one is a novel histone.  I've looked at the QIAGEN QProteome
> > Nuclear kit, but I'm loath to commit to using that as it doesn't tell
> > you what any of the buffers are.  I can't even tell if it's an acid
> > extraction or a salt extraction!  Also, it's very expensive for the
> > amount of tissue you can process with it.
>
> Histones have a high positive charge, to get a reasonable estimate of
> their molecular mass from PAGE, use the positive detergent CTAB rather
> than the negatively charged SDS (E. Buxbaum: Anal. Biochem. 314 {2003}
> 70-76)
>
> > So I'd prefer to do my own histone extraction if possible.  I believe
> > that means I want to prepare purified nuclei and then do an acid
> > extraction. Previously I've done nuclear preps when preparing high
> > molecular weight gDNA - will the same buffers be appropriate for protein
> > work?  Protocol is as follows:
> >
> > Homogenise tissue at low speed in lysis buffer [0.05M TrisCl pH 7.5, 1mM
> > EDTA, 5mM MgCl2, 50mM NaCl, 5% glycerol, 1% Triton X-100, 1% B-ME],
> > using 100 mg tissue / ml buffer.
>
> I'd probably use KCl rather than NaCl, as the cell interior is high in K
> and low in Na. I'd also reduce [Mg] to 1 mM or so to discourage
> metal-dependent proteases. The Triton I'd leave out at this step and
> reduce bME to 1 mM (or replace it with DTT, which has a more appropriate
> redox potential). You'll need a protease inhibitor coctail (EDTA, PMSF,
> Pepstatin, Leupeptin, e-aminocaproic acid).
>
> > Once I've got the purified nuclei, the histone prep looks admirably
> > straightforward.  According to Abcam, you just resuspend the nuclear
> > pellet in 0.2M HCl and leave it overnight at 4 degrees C.  Centrifuge to
> > spin down debris and keep the supernatant.  However, other protocols
> > seem to use different acid concentrations, or H2SO4 instead of HCl, and
> > may even include B-ME in the histone extraction buffer.
>
> The acid is quite irrelevant, the idea is simply to increase the
> solubility of the highly charged histons and to pellet as many of the
> other proteins as possible. The bME protects cysteine -SH groups from
> oxidation by air. However, this extraction procedure is too selective for
> your purposes, you will not know whether there are cross-reacting proteins
> in the pellet.
> >
> > One protocol I Googled called for you to neutralise the preparation with
> > KOH afterwards - is that really called for?  Wouldn't it just drop the
> > proteins straight back out of solution again?
>
> No. But you need a pH near neutral and as low an ion concentration as
> possible for the electrophoresis step. Therefore solubilisation in sample
> buffer rather than HCl is better. That also gives you an idea about the
> cross-reactions of your antibody.
>
> >  For a histone (size ~17 kDa), it'll be quite a high percentage gel.
>
> I find gradient gels (5-15 or 20%) most convenient
>
> >
> > Protein 1:   mgclh
> > ------------------
> >
> > RIPA-insoluble, so it's likely mgclh will also be RIPA-insoluble.
> > Protein size is expected to be around 55 kDa.
>
> Again the use of CTAB electrophoresis may help, as CTAB can solubilise
> proteins very efficiently.
>
> > The original literature on Gmcl1 said they resuspended the
> > RIPA-insoluble pellet in "SDS-polyacrylamide gel electrophoresis sample
> > buffer", but doesn't give the composition.  I would assume this is 1x
> > Laemmli sample buffer.  Does this sound plausible?  If so, what recipe
> > for Laemmli buffer would you recommend?  I've found half a dozen
> > different recipes, and the only common factor between them is ~60-100mM
> > Tris pH 6.8 and 2% SDS. There are variants with and without DTT, with
> > and without B-ME, with and without loading dye, using sucrose or
> > glycerol for increased density!
>
> The buffer is actually double concentrated, once you mix it with an equal
> volume of sample you get the working strenght. DTT and bME reduce
> disulphide bonds and thus lead to more complete unfolding of the proteins.
> DTT (Clelands reagent) smells less and has a better standard redox
> potential for this purpose. However, it is more expensive than bME. Wether
> you use glycerol or sucrose to increase density really doesn't matter, you
> just need either for proper gel loading, its physics, not chemistry. The
> dye is important not only to aid in loading, but even more as front
> marker, without it you can't calculate Rf-values which you need for
> molecular mass determination.
>
> > I would imagine I'll need to add B-ME to reduce the proteins before
> > running on the gel, but should I add the B-ME at the point I resuspend
> > the pellet, or only when I'm about to run an aliquot on a gel?  Will I
> > need to add protease inhibitors, or will the Laemmli buffer itself take
> > care of that?
>
> Reducing agent is part of the 2x concentrate, usually the sample/buffer
> mixture is heated to get more efficient reduction. Many people do 2 min in
> a boiling water bath and for your purposes that is probably fine. With
> large proteins I found that 10 min at 60 degrees works better. Very few
> proteases work in hot SDS under reducing conditions, but there have been
> rare cases reported where that was a problem. The protease inhibitors
> should of course be present during the homogenisation of the cells and the
> centrifugation steps. Many (like PMSF) actually chemically inactivate
> proteases, solving the problem permanently anyway.
>
>
> ------------------------------
>
> Message: 6
> Date: Wed, 18 Jun 2008 15:51:44 GMT
> From: dk from no.email.thankstospam.net (DK)
> Subject: E.coli strain recommendation, please
> To: methods from net.bio.net
> Message-ID: <g3batr$236$2 from news.albasani.net>
>
> Traditionally, for reasons I am not quite sure about, we use
> Invitrogen's TOP10 cells for all routine cloning. There is one
> serious problem with this strain, however. Cells grow too slowly.
> E.g. to get a decent size clone, plates need to be incubated
> 24 hours. In a typical overnight (4 pm - 9 am), clones are too
> small.  Also, "short" overnight liquid cultures are not saturated,
> resulting in low plasmid yields. This is becoming a problem
> when we want to work fast and not waste time waiting for the
> bugs to grow. .
>
> So I am looking for alternatives that offer:
>
> 1. High transformation efficiency (both chemical
> and electroporation).
> 2. Low level of recombination.
> 3. High plasmid yields and quality of DNA minipreps.
> 4. Fast growth.
>
> TOP10 fit requirements #1-2, #3 if we wait long enough
> but definitely not #4.
>
> Thanks,
>
> Dima
>
>
>
> ------------------------------
>
> Message: 7
> Date: Wed, 18 Jun 2008 21:32:31 +0530
> From: "priyadarsini Tripuraneni" <priyatripuraneni from gmail.com>
> Subject: doubt regarding agrobacterium DNA restriction digestion
> To: methods from magpie.bio.indiana.edu
> Message-ID:
>        <e67ee9730806180902lec5f29ftee91fffe48cb4a06 from mail.gmail.com>
> Content-Type: text/plain; charset=ISO-8859-1
>
> hi all
> as i am attempting to digest agrobacterium DNA i am not able to do that.
> please help me regarding this.
>
> --
> prasanthi
>
>
> ------------------------------
>
> Message: 8
> Date: Wed, 18 Jun 2008 08:57:27 -0700 (PDT)
> From: Nick Theodorakis <nick.theodorakis from gmail.com>
> Subject: Re: E.coli strain recommendation, please
> To: methods from net.bio.net
> Message-ID:
>        <641384b3-ff96-4c1b-a104-6d343b539ab8 from 26g2000hsk.googlegroups.com>
> Content-Type: text/plain; charset=ISO-8859-1
>
> On Jun 18, 11:51 am, d... from no.email.thankstospam.net (DK) wrote:
> > Traditionally, for reasons I am not quite sure about, we use
> > Invitrogen's TOP10 cells for all routine cloning. There is one
> > serious problem with this strain, however. Cells grow too slowly.
> > E.g. to get a decent size clone, plates need to be incubated
> > 24 hours. In a typical overnight (4 pm - 9 am), clones are too
> > small.  Also, "short" overnight liquid cultures are not saturated,
> > resulting in low plasmid yields. This is becoming a problem
> > when we want to work fast and not waste time waiting for the
> > bugs to grow. .
> >
> > So I am looking for alternatives that offer:
> >
> > 1. High transformation efficiency (both chemical
> > and electroporation).
> > 2. Low level of recombination.
> > 3. High plasmid yields and quality of DNA minipreps.
> > 4. Fast growth.
> >
>
> I like DH5alpha for my routine subcloning and plasmid prep needs.
>
> Nick
>
> --
> Nick Theodorakis
> nick_theodorakis from hotmail.com
> contact form:
> http://theodorakis.net/contact.html
>
>
> ------------------------------
>
> _______________________________________________
> Methods mailing list
> Methods from net.bio.net
> http://www.bio.net/biomail/listinfo/methods
>
> End of Methods Digest, Vol 37, Issue 14
> ***************************************
>


-- 
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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