3 questions

Jim Young via methods%40net.bio.net (by jyyoungjimmy from gmail.com)
Thu May 29 10:05:45 EST 2008


1. It is possible that you have two bands using poly and mono because you
protein might be degraded and your antibodies recognize different epitopes.
2. if you use the marker, size should be the same. Another possibility is
the degraded proteins.
3. It is difficult to quantify your protein by Western. You have to have
detailed control, such as BSA or other quantified tag protein.
4. If you use Western a lot, you should use this kit:
http://www.genscript.com/western_tech.html?src=forum
You can finish western in a hour. The advanced kit is very sensitive but
usually I use the complete kit:
http://www.genscript.com/western_complete_kit.html?src=forum



On Fri, May 23, 2008 at 1:03 PM, <methods-request from oat.bio.indiana.edu>
wrote:

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> Today's Topics:
>
>   1. Re: qRT-PCR and DNA contamination (Jeremy Coate)
>   2. 3 questions (Kamalian, Laleh)
>   3. Re: IP Protocols and hints (Nick Theodorakis)
>   4. Re: Hydroshear DNA (Nick Theodorakis)
>   5. Re: Hydroshear DNA (peter)
>   6. Hydroshear DNA (peter)
>
>
> ----------------------------------------------------------------------
>
> Message: 1
> Date: Fri, 23 May 2008 05:55:14 -0400
> From: "Jeremy Coate" <jec73 from cornell.edu>
> Subject: Re: qRT-PCR and DNA contamination
> To: methods from oat.bio.indiana.edu
> Message-ID:
>        <42f327750805230255w15d3bd0co269eea50747b33d9 from mail.gmail.com>
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>
> I would reiterate a point that Brian made earlier in this string - negative
> controls frequently exhibit some "amplification" in qPCR, but if the cycle
> number is high it should not be a concern.
>
> Even if you do have some residual genomic contamination that is amplifying
> in the absence of cDNA (I'm guessing you are running controls in which you
> do not reverse transcribe your RNA), it probably is not contributing to the
> signal in your cDNA samples. My no-RT controls frequently show some
> amplification (at high Ct's) that does indeed appear to be from genomic
> DNA,
> but when I run the cDNA reactions on a gel there is no evidence of genomic
> amplifcation. Presumably this is because the cDNA is in much greater
> abundance than the residual genomic contamination. If the Ct of your
> negative control is 10 cycles higher than your cDNA samples then the
> genomic
> DNA is only about 0.1% of the cDNA abundance.
>
> And, as Brian said, you frequently get a Ct value even if there is no DNA
> template - its not unusual for me to see Ct's of 33 or so for my water
> controls. But as long as your target templates are amplifying at much lower
> cycles, whatever this artifactual amplifcation is should not be affecting
> your results in any significant way. Of course, if you are getting
> amplification in your controls at cycles similar to your targets, then you
> do have a significant contamination problem, and the other suggestions
> posted here apply.
>
> Jeremy
>
> On Thu, May 22, 2008 at 4:19 PM, Analia Alet <analia_alet from intech.gov.ar>
> wrote:
>
> > Hi Emad, for DNAse treatment I have employed TURBO DNA Free form Ambion
> > (cat #
> > 1907) and it worked well for me. Perhaps your problem is not the DNase.
> > Perhaps you have two many DNA/ ug RNA. This kit, for example, removes up
> to
> > 50
> > ug DNA/ml RNA. If there is mor DNA in the sample, the manufactures advise
> > to
> > do a rigorous DNAse treatment, were you use more DNAse, adding it in to
> > steps
> > to the reaction mix and you duplicate the reaction time.
> >
> > Another thing to wonder: are you DNAse working well?
> > Also you can optimize the digestion times with your owun sample and your
> > own
> > kit. Perhaps you need to treat the samples for more time. Remember that
> > kits
> > are standars, and sometimes you have to adjust them to your own
> conditions.
> >
> > Here it is the protocol we used:
> >
> > Reaction mix:
> > 20ug RNA
> > 10ul 10X Buffer
> > 2ul  TURBO DNA-free
> > up to 100 ul  water
> >
> > incubate 30 min 37ºC
> > add 10 ul DNAse inactivation reagent
> > incubate 2 min at room temperature (mix 2-3 times by inversion)
> > ccentrifugate 15 min 10000g 4ºC
> > SN to a new tube
> > Store -80ºC
> >
> > Hope it works for you
> > Good luck
> > Best regards
> > Analía
> >
> >
> >
> >
> > Mensaje citado por methods-request from oat.bio.indiana.edu:
> >  Hello,
> >
> > I am doing real time PCR for analysing my genes expression under
> different
> > condition of my fungus. We use SYPR green. Actually I isolated RNA using
> > both kits Qiagen and Peqlab (Germany) and treated the RNA with DNase on
> > column following the kit's instruction. Unfortunately I could still
> detect
> > a
> > DNA contamination!! I treated the RNA again with DNase from NEB but still
> > some traces of DNA exist. I tried to avoid the DNase treatment because I
> > learned from some people who deal with RT-PCR to avoid DNase treatment
> and
> > used Intron Span primers, and here is the big suffering, I used 18-20mere
> > primers which locate just in the middle of exon borders and still these
> > primers can bind the pure DNA, I reduced to 6 bases on the 3'-exon and
> > still
> > get some amplification, isn't that terrible? I thought to go to 3 bases
> at
> > the 3' end of the primer but then you don't get any good prime which suit
> > in
> > this area!!?
> >
> > Can somebody please give me some hints or has any experience in such a
> good
> > DNase which doesn't leave any DNA traces?! I am not optimistic in that.
> >
> > Emad
> >
> > _______________________________________________
> > Methods mailing list
> > Methods from net.bio.net
> > http://www.bio.net/biomail/listinfo/methods
> >
>
>
>
> --
> Jeremy Coate
> Dept. of Plant Biology
> Cornell University
> jec73 from cornell.edu
> 607-342-2679
>
>
> ------------------------------
>
> Message: 2
> Date: Fri, 23 May 2008 11:17:35 +0100
> From: "Kamalian, Laleh" <L.Kamalian from liverpool.ac.uk>
> Subject: 3 questions
> To: "methods" <methods from magpie.bio.indiana.edu>
> Message-ID:
>        <31DC2DFFD70C174B8E3072803F894F91046C89 from EVSSTAFF1.livad.liv.ac.uk>
> Content-Type: text/plain;       charset="iso-8859-1"
>
> I have 3 questions if someone could help me with those please.
> 1. Could 2 different bands (size wise) be detected for the same protein
> using poly colonal and monoclonal antibodies?
> 2.  If the samples are run on electrophoresis for too long, can the size of
> the protein show difference comparing to the previous (shorter) runs?
> 3.  In siRNA , I have knock down my gene of interest at the mRNA level
> (70-80% according to quantitative PCR x2 experiments), but western blot of
> the same colony cell protein lysates show no or less ( only 30% ) decrement.
>  Any explanation please?
>
> Thanks
> Laleh
>
>
> ------------------------------
>
> Message: 3
> Date: Fri, 23 May 2008 06:45:51 -0700 (PDT)
> From: Nick Theodorakis <nick.theodorakis from gmail.com>
> Subject: Re: IP Protocols and hints
> To: methods from net.bio.net
> Message-ID:
>        <532739c6-c058-4830-8612-f6db5ab536c2 from x35g2000hsb.googlegroups.com>
> Content-Type: text/plain; charset=ISO-8859-1
>
> On May 23, 2:44 am, Bean Long <ben.l... from yourfinger.anu.edu.au> wrote:
>
>
> >
> > Many thanks AK. I s'pose 'enrich' would be a better word? The hope is to
> > do a bit of proteomic work and subunit relative abundance analysis on
> > it. Enough to run relative pure stuff on a gel would be handy. I had a
> > little play with the Protein G-sepharsoe I found but ended up with
> > bucket loads of what I assume is Protein G on my gels. I also think it's
> > a little too old for successful IP, so I'm getting a new batch!
>
> More likely it was IgG you saw on your gel. The heavy chains are
> between 50-60 kDa and the light chains are 20-something-ish kDa.
>
> Nick
>
> --
> Nick Theodorakis
> nick_theodorakis from hotmail.com
> contact form:
> http://theodorakis.net/contact.html
>
>
> ------------------------------
>
> Message: 4
> Date: Fri, 23 May 2008 06:47:06 -0700 (PDT)
> From: Nick Theodorakis <nick.theodorakis from gmail.com>
> Subject: Re: Hydroshear DNA
> To: methods from net.bio.net
> Message-ID:
>        <712c8ee2-2d34-4b8a-b774-bcc7e08dc336 from c65g2000hsa.googlegroups.com>
> Content-Type: text/plain; charset=ISO-8859-1
>
> On May 23, 8:34 am, peter <peter.ianak... from gmail.com> wrote:
> > Hello guys,
> > I need some expertise in random DNA fragmentation. I was thinking of
> > hydroshearing DNA by passing trough syringe. Does anyone know the
> > diameter of the needle needed to shear the DNA? is there better
> > methods? - any experience is greatly appreciated.
> > Thanks,
> > Peter
>
> How big do you need the pieces to be and what size distribution is
> acceptable?
>
> Nick
>
> --
> Nick Theodorakis
> nick_theodorakis from hotmail.com
> contact form:
> http://theodorakis.net/contact.html
>
>
> ------------------------------
>
> Message: 5
> Date: Fri, 23 May 2008 06:56:44 -0700 (PDT)
> From: peter <peter.ianakiev from gmail.com>
> Subject: Re: Hydroshear DNA
> To: methods from net.bio.net
> Message-ID:
>        <c782356c-1f99-4cb7-8978-3d90cbae1ff8 from w7g2000hsa.googlegroups.com>
> Content-Type: text/plain; charset=ISO-8859-1
>
> On May 23, 9:47 am, Nick Theodorakis <nick.theodora... from gmail.com>
> wrote:
> > On May 23, 8:34 am, peter <peter.ianak... from gmail.com> wrote:
> >
> > > Hello guys,
> > > I need some expertise in random DNA fragmentation. I was thinking of
> > > hydroshearing DNA by passing trough syringe. Does anyone know the
> > > diameter of the needle needed to shear the DNA? is there better
> > > methods? - any experience is greatly appreciated.
> > > Thanks,
> > > Peter
> >
> > How big do you need the pieces to be and what size distribution is
> > acceptable?
> >
> > Nick
> >
> > --
> > Nick Theodorakis
> > nick_theodora... from hotmail.com
> > contact form:http://theodorakis.net/contact.html
>
> The distribution is not that important , just I need complete random
> representation. So far I am doin low complexity libraries, when I go
> to human DNA, then I will have to get it into the tight range say 0.5
> to 2 kb.
> -Peter
>
>
> ------------------------------
>
> Message: 6
> Date: Fri, 23 May 2008 05:34:40 -0700 (PDT)
> From: peter <peter.ianakiev from gmail.com>
> Subject: Hydroshear DNA
> To: methods from net.bio.net
> Message-ID:
>        <5aa1d425-dcae-4ab1-8a6a-4b3d6a0a7e7b from k13g2000hse.googlegroups.com>
> Content-Type: text/plain; charset=ISO-8859-1
>
> Hello guys,
> I need some expertise in random DNA fragmentation. I was thinking of
> hydroshearing DNA by passing trough syringe. Does anyone know the
> diameter of the needle needed to shear the DNA? is there better
> methods? - any experience is greatly appreciated.
> Thanks,
> Peter
>
>
> ------------------------------
>
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