Methods Digest, Vol 33, Issue 26

Virash Gupta via methods%40net.bio.net (by virashkgupta from gmail.com)
Tue Feb 26 04:38:12 EST 2008


Besides all above since you are getting right amplification but weak, try
decreasing dNTP concentration to one half and one fourth the one you have
been using. I have experienced strong inhibitions in specific PCr
ampifications only due to use of higher dNTP concentrations only.

On 2/25/08, methods-request from oat.bio.indiana.edu <
methods-request from oat.bio.indiana.edu> wrote:
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> Today's Topics:
>
>   1. Re: pcr prob (ChenHA)
>   2. Re: pcr prob (chovek69)
>   3. Re: pcr prob (Aawara Chowdhury)
>   4. Re: pcr prob (ChenHA)
>
>
> ----------------------------------------------------------------------
>
> Message: 1
> Date: Sun, 24 Feb 2008 13:40:53 +0000
> From: ChenHA <hzhen from freeuk.com>
> Subject: Re: pcr prob
> To: methods from net.bio.net
> Message-ID: <1203860754.14229.0 from proxy00.news.clara.net>
> Content-Type: text/plain; charset=ISO-8859-1; format=flowed
>
> harold b wrote:
> > Hi, my negative control (no template with both primers and mastermix)
> > consistently shows a stronger single band whereas my amplicons show a
> > leading similar but weaker band followed by a fading smear reminiscent
> > of non-amplified DNA. In each case the product with DNA is less
> > intense. Can someone make sense of this?
>
> The strong band is most likely just your primers.  Easy to check by
> running the same amount of primers used on the gel.  The weaker band is
> most likely the desired PCR product, check the size of the band (run
> marker), the smear may be non-specific amplification products.  If the
> weaker band is the right size, then you got what you wanted, you can
> just cut it out if you want to use it for cloning if that is what you
> want to do.
>
> If you want to use the PCR  for pretty picture, then think about
> increasing the number of cycles, changing (most probably by increasing)
> the annealing temperature, changing the Mg++ concentration, lowering the
> amount of primers, etc.   Don't bother with redesigning the primer
> unless you got the wrong PCR product.
>
>
>
>
> ------------------------------
>
> Message: 2
> Date: Sun, 24 Feb 2008 02:39:44 -0800 (PST)
> From: chovek69 <ivanoov from gmail.com>
> Subject: Re: pcr prob
> To: methods from net.bio.net
> Message-ID:
>        <c4c4ec4f-9956-4975-8558-a9333f8b17ee from z70g2000hsb.googlegroups.com>
> Content-Type: text/plain; charset=ISO-8859-1
>
> Most probably your bands in the negative controls are actually primer
> dimers. How big is the expected band ?  The dimers are weaker in the
> DNA samples because some of the primers are used for the expected
> product. I feel that you need to re-design primers or do extensive PCR
> optimization.
>
> Regards
> Ivan
>
>
> On Feb 23, 6:20 pm, harold  b <theoha... from gmail.com> wrote:
> > Hi, my negative control (no template with both primers and mastermix)
> > consistently shows a stronger single band whereas my amplicons show a
> > leading similar but weaker band followed by a fading smear reminiscent
> > of non-amplified DNA. In each case the product with DNA is less
> > intense. Can someone make sense of this?
>
>
>
> ------------------------------
>
> Message: 3
> Date: Sun, 24 Feb 2008 20:46:21 GMT
> From: Aawara Chowdhury <aawara from pontiff-playground.org>
> Subject: Re: pcr prob
> To: methods from net.bio.net
> Message-ID: <xIkwj.9603$0M3.8182 from newsfe17.lga>
>
> In <1203860754.14229.0 from proxy00.news.clara.net>,
> ChenHA <hzhen from freeuk.com> wrote:
>
> > harold b wrote:
> >> Hi, my negative control (no template with both primers and mastermix)
> >> consistently shows a stronger single band whereas my amplicons show a
> >> leading similar but weaker band followed by a fading smear reminiscent
> >> of non-amplified DNA. In each case the product with DNA is less
> >> intense. Can someone make sense of this?
> >
> > The strong band is most likely just your primers.  Easy to check by
> > running the same amount of primers used on the gel.
>
> The strong band is unlikely to be "just your primers".  As someone
> earlier in this thread described, the strong band is what has
> quixotically been labeled as "primer dimer" - the result of DNA synthesis
> after a primer anneals (partially) to itself or the other primer in the
> PCR.
>
> Loading 5 or 10 picomoles of each primer on an agarose gel by itself
> will not give a band that fluoresces brightly in the presence of
> ethidium bromide.
>
> AC
> --
> Email: echo 36434455860060025978157675027927670979097959886449930P | dc
>
>
> ------------------------------
>
> Message: 4
> Date: Sun, 24 Feb 2008 22:45:33 +0000
> From: ChenHA <hzhen from freeuk.com>
> Subject: Re: pcr prob
> To: methods from net.bio.net
> Message-ID: <1203893436.24100.0 from proxy01.news.clara.net>
> Content-Type: text/plain; charset=ISO-8859-1; format=flowed
>
> Aawara Chowdhury wrote:
> > In <1203860754.14229.0 from proxy00.news.clara.net>,
> >  ChenHA <hzhen from freeuk.com> wrote:
> >
> > The strong band is unlikely to be "just your primers".  As someone
> > earlier in this thread described, the strong band is what has
> > quixotically been labeled as "primer dimer" - the result of DNA
> synthesis
> > after a primer anneals (partially) to itself or the other primer in the
> PCR.
> >
> > Loading 5 or 10 picomoles of each primer on an agarose gel by itself
> > will not give a band that fluoresces brightly in the presence of
> > ethidium bromide.
> >
>
> How do you know how much primers the original poster added?  It is for
> the original poster to comfirm or deny, or check by runnning the gel (as
> I stated, by using the same amount), not for you to assert.  Whoever
> gives you the idea the 5 or 10 picomoles is what everyone uses?  I
> don't, and I have done perhaps thousands of successful PCR reactions.
> And they do fluoresces, how brightly of course depends on how much you
> add.
>
> As it happens, I see this kind of things quite often, from my own PCR.
>
>
> > AC
>
>
> ------------------------------
>
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> End of Methods Digest, Vol 33, Issue 26
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-- 
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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