Methods Digest, Vol 33, Issue 27

Virash Gupta via methods%40net.bio.net (by virashkgupta from gmail.com)
Tue Feb 26 10:47:56 EST 2008


Subject: PCR problem
Theory of PCR is simple to understand but sometimes difficult to practise.
Normally such problems should not come but are routenly encountered. I will
lay stess on only two points. First, you are getting right amplification-
that is good at least primer is working. Second, problem is with - Primer
dimer- this happens when primers are not utilized in amplification due to
low amplification. Under normally use conditions right primer must amplify
normally and in sufficient amount. Here you are getting weak amplification
with smearing. one of the most seen cause for low amplification is use of
higher conc of dNTPs- not known to many.  Lower amount of dNTP also blocks
amplification due to nonavailability of enough building blocks. Just try
with 2-fold and half conc of dNTP- I am sure it will enhance amplification.
Regarding smearing it is normally due to your dNTPS/ primers/ water being
contaminated with endonucleases which degrades the template in initial step=
s
to reduce its concentration. Try with every thing fresh. You can also try
with purified DNA (reprecipitation), freshly diluted primer mix, dNTPs all
with freshly sterilized D water. Work optimistically. I am sure you are not
using home made taq. All the best
Virash
On Mon, Feb 25, 2008 at 11:20 PM, <methods-request from oat.bio.indiana.edu>
wrote:

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> Today's Topics:
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>   1. Re: Methods Digest, Vol 33, Issue 25 pcr prob (rijuta kotenkar)
>   2. Re: pcr prob (DK)
>   3. Re: pcr prob (Sudheendra Rao N R)
>   4. Re: pcr prob (Aawara Chowdhury)
>   5. Re: pcr prob (Aawara Chowdhury)
>   6. Re: pcr prob (Aawara Chowdhury)
>   7. Re: pcr prob (Aawara Chowdhury)
>   8. Re: pcr prob (ChenHA)
>   9. Re: pcr prob (Aawara Chowdhury)
>
>
> ----------------------------------------------------------------------
>
> Message: 1
> Date: Mon, 25 Feb 2008 08:19:20 +0100
> From: "rijuta kotenkar" <rijutak from googlemail.com>
> Subject: Re: Methods Digest, Vol 33, Issue 25 pcr prob
> To: methods from oat.bio.indiana.edu
> Message-ID:
>        <71d8f0270802242319n7b75500ala0d077741d3cde4f from mail.gmail.com>
> Content-Type: text/plain; charset=3DWINDOWS-1252
>
> Hi!
> Did you check if your primers form Primer-dimer?? Or check if your primer
> has been contaminated by the template.
>
> Rijuta
>
> On Sun, Feb 24, 2008 at 6:03 PM, <methods-request from oat.bio.indiana.edu>
> wrote:
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> >
> >
> > Today's Topics:
> >
> >   1. pcr prob (harold b)
> >
> >
> > ----------------------------------------------------------------------
> >
> > Message: 1
> > Date: Sat, 23 Feb 2008 08:20:09 -0800 (PST)
> > From: harold  b <theoharis from gmail.com>
> > Subject: pcr prob
> > To: methods from net.bio.net
> > Message-ID:
> >        <74fec1a4-d732-49a4-9414-8b9920f4ce94 from s8g2000prg.googlegroups.co=
m
> >
> > Content-Type: text/plain; charset=3DISO-8859-1
> >
> > 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?
> >
> >
> > ------------------------------
> >
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> > End of Methods Digest, Vol 33, Issue 25
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> >
>
>
>
> --
> "In the arithmetic of love, one plus one equals everything, and two minus
> one equals nothing=85."- Mignon Mc Laughlin
>
>
> ------------------------------
>
> Message: 2
> Date: Mon, 25 Feb 2008 14:06:47 GMT
> From: dk from no.email.thankstospam.net (DK)
> Subject: Re: pcr prob
> To: methods from net.bio.net
> Message-ID: <TXzwj.101$Td5.25 from newsfe07.lga>
>
> In article <qlywj.9872$0M3.986 from newsfe17.lga>,
> aawara from pontiff-playground.org wrote:
> >In <1203893436.24100.0 from proxy01.news.clara.net>,
> > ChenHA <hzhen from freeuk.com> wrote:
> >
> >> How do you know how much primers the original poster added?
> >
> >The amount doesn't matter - see below.
> >
> >> 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.
> >
> >Ethidium bromide binds single-stranded DNA extremely poorly, if at all.
> >It fluoresces when bound to RNA, simply because most RNA molecules do
> >fold-back upon themselves to form partial duplexes.  Long ssDNAs also
> >fluoresce in the presence of ethidium for the same reason.
> >
> >I find it highly improbably that your ss oligonucleotides snap back on
> >themselves sufficiently to fluoresce with ethidium, in the absence of
> >polymerization that makes them ds.
>
> FWIW, every time my PCR did not work or produced low yield,
> a diffuse and very weakly stained band below 100 bp can be seen.
> I always interpreted it as "unused primers". Granted, typically the
> amount loaded on the gel would correspond to < 50 ng and it is
> doubtful that EthBr has this sensitivity for ss DNA. Still, the fact
> is that whatever the primers do (dimers, hairpins) they do
> bind EthBr.
>
> DK
>
>
>
>
>
>
> ------------------------------
>
> Message: 3
> Date: Mon, 25 Feb 2008 09:43:10 +0530
> From: "Sudheendra Rao N R" <sudhee26 from gmail.com>
> Subject: Re: pcr prob
> To: "harold b" <theoharis from gmail.com>, methods
>        <Methods from magpie.bio.indiana.edu>
> Message-ID:
>        <a1a1abbb0802242013t60e3c22egbf3d1270fa5f3621 from mail.gmail.com>
> Content-Type: text/plain; charset=3DISO-8859-1
>
> Hi,
> What is your reaction mix volume?
> What is your desired pcr product size?
> Primer dimers are more likely in 10 microliter pcr reaction mix.
> Also they tend to be below 150bp.
> Increase reaction volume to 25 microlitre atleast.
> Calculate annealing temperature using wallace rule and use that (though i=
t
> is Td but it helps better for primers about 20 bp length).
> Put primer only 0.2-0.4microM in total reaction mix.
>
> i guess that will help.
> Sudheendra.
>
> On Sat, Feb 23, 2008 at 9:50 PM, harold b <theoharis 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?
> > _______________________________________________
> > Methods mailing list
> > Methods from net.bio.net
> > http://www.bio.net/biomail/listinfo/methods
> >
>
>
>
> --
> Think before agree
> Think before you nod
> but STOP thinking
> and You Are God
>
>
> ------------------------------
>
> Message: 4
> Date: Mon, 25 Feb 2008 12:17:26 GMT
> From: Aawara Chowdhury <aawara from pontiff-playground.org>
> Subject: Re: pcr prob
> To: methods from net.bio.net
> Message-ID: <qlywj.9872$0M3.986 from newsfe17.lga>
>
> In <1203893436.24100.0 from proxy01.news.clara.net>,
>  ChenHA <hzhen from freeuk.com> wrote:
>
> > How do you know how much primers the original poster added?
>
> The amount doesn't matter - see below.
>
> > It is for
> > the original poster to comfirm or deny, or check by runnning the gel (a=
s
> > 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.
>
> Ethidium bromide binds single-stranded DNA extremely poorly, if at all.
> It fluoresces when bound to RNA, simply because most RNA molecules do
> fold-back upon themselves to form partial duplexes.  Long ssDNAs also
> fluoresce in the presence of ethidium for the same reason.
>
> I find it highly improbably that your ss oligonucleotides snap back on
> themselves sufficiently to fluoresce with ethidium, in the absence of
> polymerization that makes them ds.
>
> AC
> --
> Email: echo 36434455860060025978157675027927670979097959886449930P | dc
>
>
> ------------------------------
>
> Message: 5
> Date: Mon, 25 Feb 2008 12:32:54 GMT
> From: Aawara Chowdhury <aawara from pontiff-playground.org>
> Subject: Re: pcr prob
> To: methods from net.bio.net
> Message-ID: <Wzywj.9874$0M3.1972 from newsfe17.lga>
>
> In <qlywj.9872$0M3.986 from newsfe17.lga>,
>  Aawara Chowdhury <aawara from pontiff-playground.org> wrote:
>
> > In <1203893436.24100.0 from proxy01.news.clara.net>,
> >  ChenHA <hzhen from freeuk.com> wrote:
> >
> >> How do you know how much primers the original poster added?
> >
> > The amount doesn't matter - see below.
> >
> >> 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.
> >
> > Ethidium bromide binds single-stranded DNA extremely poorly, if at all.
> > It fluoresces when bound to RNA, simply because most RNA molecules do
> > fold-back upon themselves to form partial duplexes.  Long ssDNAs also
> > fluoresce in the presence of ethidium for the same reason.
> >
> > I find it highly improbable that your ss oligonucleotides snap back on
> > themselves sufficiently to fluoresce with ethidium, in the absence of
> > polymerization that makes them ds.
>
> BTW, there are several good papers on why ethidium fluoresces when bound
> to double-stranded nucleic acids, but not single-stranded ones.  Here
> are two:
>
> Decay of Fluorescence Emission Anisotropy of the Ethidium Bromide-DNA
> Complex Evidence for an Internal Motion in DNA.  Wahl, Paoletti, & Le Pec=
q
> PNAS 65:417-421 (1970).
>
> Ethidium Bromide Does Not Fluoresce when Intercalated Adjacent to
> 7-Deazaguanine in Duplex DNA.  Latimer & Lee. JBC 266:13849-51 (1991).
>
> The latter has several citation which examine in some depth the
> mechanisms by which ethidium bromide fluoresces when bound to nucleic
> acids.
>
> AC
> --
> Email: echo 36434455860060025978157675027927670979097959886449930P | dc
>
>
> ------------------------------
>
> Message: 6
> Date: Mon, 25 Feb 2008 14:01:01 GMT
> From: Aawara Chowdhury <aawara from pontiff-playground.org>
> Subject: Re: pcr prob
> To: methods from net.bio.net
> Message-ID: <xSzwj.58285$Ft5.57023 from newsfe15.lga>
>
> In <1203946852.17846.0 from proxy01.news.clara.net>,
>  ChenHA <hzhen from freeuk.com> wrote:
>
> > Aawara Chowdhury wrote:
> >
> >> P.S. You don't have to be so rude, just because someone points out
> >> an incongruence in your "observation".
> >>
> >
> > Geez, now you are truly stupid.  Why put the quotation marks on
> > observation?  Are you suggesting that observations were wrong, or false=
,
> > or I have mistaken a reflection of a light bulb to be a band?  What's
> > incongruent about my observation?  Observations are observations,
> > science comes out of observation, idiot.  I have shown that you made
> > unwarranted assumption on primer concentration and length, and you stil=
l
> > insist on piling on casting doubt on my "observation".  Stupid people
> > deserve all the insult they get.
>
> Your "observation" is incongruent with everything that has been published
> about the function of ethidium bromide.  You "observe" that ethidium
> fluoresces "brightly" when bound to single-stranded oligonucleotides.
> That either means that your oligonucleotides were not single-stranded
> (odd for PCR primers), or that there's an error with your "observation".
> And yes, that is precisely why I find your "observation" perplexing.
> And unfortunately for you, science is not just about making an
> "observation";
> it also requires being prepared to subject an observation to review.
>
> And my, don't we like calling people stupid.  Makes you feel intelligent,
> does it?
>
> AC
> --
> Email: echo 36434455860060025978157675027927670979097959886449930P | dc
>
>
> ------------------------------
>
> Message: 7
> Date: Mon, 25 Feb 2008 14:25:25 GMT
> From: Aawara Chowdhury <aawara from pontiff-playground.org>
> Subject: Re: pcr prob
> To: methods from net.bio.net
> Message-ID: <pdAwj.58287$Ft5.57384 from newsfe15.lga>
>
> In <TXzwj.101$Td5.25 from newsfe07.lga>,
>  DK <dk from no.email.thankstospam.net> wrote:
>
> > FWIW, every time my PCR did not work or produced low yield,
> > a diffuse and very weakly stained band below 100 bp can be seen.
> > I always interpreted it as "unused primers". Granted, typically the
> > amount loaded on the gel would correspond to < 50 ng and it is
> > doubtful that EthBr has this sensitivity for ss DNA. Still, the fact
> > is that whatever the primers do (dimers, hairpins) they do
> > bind EthBr.
>
> Dima - agreed completely.  But your PCRs contain polymerase and dNTPs;
> so your "unused primers", are in fact the primer-dimer products that
> are the result of primers annealing to themselves, and permitting
> the synthesis of dsDNA (that binds ethidium) in the presence of dNTPs
> and polymerase.
>
> Try doing this same reaction without polymerase, you will not see
> the fluorescent band (or without dNTPs for that matter).
>
> Ethidium requires base-stacking to fluoresce.  It doesn't intercalate
> into adjacent base-pairs because of steric hindrance.  And a minimum
> of three stacked ethidium molecules are needed for fluorescence to
> be observed.  Thus the minimal ds nucleic acid that does fluoresce
> with ethidium is 6 bp in length (work published by someone who used
> to be UW-Madison - Khorana).
>
> Therefore, for oligonucletides to fluoresce directly with ethidium,
> they must form at least contiguous base-pairs, a scenario that is
> certainly possible, but high unlikely for PCR primers.
>
> AC
> --
> Email: echo 36434455860060025978157675027927670979097959886449930P | dc
>
>
> ------------------------------
>
> Message: 8
> Date: Mon, 25 Feb 2008 12:59:11 +0000
> From: ChenHA <hzhen from freeuk.com>
> Subject: Re: pcr prob
> To: methods from net.bio.net
> Message-ID: <1203944654.12385.0 from proxy02.news.clara.net>
> Content-Type: text/plain; charset=3DISO-8859-1; format=3Dflowed
>
> Aawara Chowdhury wrote:
> > In <1203893436.24100.0 from proxy01.news.clara.net>,
> >  ChenHA <hzhen from freeuk.com> wrote:
> >
> >> How do you know how much primers the original poster added?
> >
> > The amount doesn't matter - see below.
> >
> >> 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.
> >
> > Ethidium bromide binds single-stranded DNA extremely poorly, if at all.
> > It fluoresces when bound to RNA, simply because most RNA molecules do
> > fold-back upon themselves to form partial duplexes.  Long ssDNAs also
> > fluoresce in the presence of ethidium for the same reason.
> >
> > I find it highly improbably that your ss oligonucleotides snap back on
> > themselves sufficiently to fluoresce with ethidium, in the absence of
> > polymerization that makes them ds.
> >
>
> Are you in the habit of teaching your grandma to suck eggs?  Perhaps I
> should have added when I said that I have seen it often before, that I
> have also checked with running the primers by itself on the gel, and saw
> a bright glowing band?  And I have even ran primers down a gel
> filtration column and see big peak of aggregated primers.  Can that the
> cause of the bright bands? I don't know because it is not something
> worth investigating further, and all I can say is that I have seen it
> and primers alone often give big bright bands.
>
> And where did you read how long the primers are that the original poster
> used?
>
> I have read the other reply by someone else suggesting primer dimer, and
> I am merely offering an alternative explanation which satisfactorily
> explain in most cases what I have seen myself, and offered a few other
> suggestions on how to proceed further (how to check and how to resolve a
> problem like this, and yes, I have seen cases where it might be primer
> dimer), and to correct the impression that anyone need to redesign the
> primer if the PCR gives the correct band (usually you just need to
> adjust a few parameters and the result can be very good).  If the
> original poster ran his primer on the gel and see no bright band, then
> he will conclude that the other poster's suggestion of primer dimer is
> correct and I am wrong, no problem.  What you have contributed is
> precisely nothing except to annoy me and showed your own stupidity.  Now
> go away.
>
>
>
> > AC
>
>
> ------------------------------
>
> Message: 9
> Date: Mon, 25 Feb 2008 15:20:45 GMT
> From: Aawara Chowdhury <aawara from pontiff-playground.org>
> Subject: Re: pcr prob
> To: methods from net.bio.net
> Message-ID: <h1Bwj.7782$f8.4340 from newsfe23.lga>
>
> In <1203951168.24775.0 from proxy01.news.clara.net>,
>  ChenHA <hzhen from freeuk.com> wrote:
>
> > Certainly from the evidence more intelligent than you.  Which bit of th=
e
> > "unwarranted assumption on primer concentration and length" don't you
> > understand?  What is the length of primers used?  You don't know (mine
> > are usually more than 30 but less than 60, very occasionally 80 or
> > more).  Do they fold back?  Possibly, or perhaps they form loops, or
> > whatever (and very probably, these have no relevance to what happened i=
n
> > hot reactions compare to what might happen in a room temperature gel).
> > Do they form primer dimer in the reaction, maybe, maybe not, but from m=
y
> > own experience they usually aren't primer dimers (and they may indeed
> > form dimer in gel, but that may have no relevance to what happened in
> > the reaction).  You are arguing from no experience, read a few things
> > then spouting off about things you have no experience of.
> > Idiot.
>
> Highly unlikely you have primers that dimerize so stably that they bind
> ethidium.  And if they did, they'd be very poor as primers for PCR.
>
> Nonethess, your name-calling doesn't remove the fact that you continue
> to claim, in the face of plenty of publications demonstrating the latter,
> that single-stranded primers bind EtBr.
>
> Wonder what your reaction is like to critiques of manuscripts, if you've
> submitted any.
>
> AC
> --
> Email: echo 36434455860060025978157675027927670979097959886449930P | dc
>
>
> ------------------------------
>
> _______________________________________________
> Methods mailing list
> Methods from net.bio.net
> http://www.bio.net/biomail/listinfo/methods
>
> End of Methods Digest, Vol 33, Issue 27
> ***************************************
>



--=20
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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