Methods Digest, Vol 64, Issue 5 NTR amplification

Virash Gupta via methods%40net.bio.net (by virashkgupta from gmail.com)
Mon Sep 20 23:04:19 EST 2010


Dear Magda,
Should not be a very serious problem as it seems. We often face such
situations. Problem of amplification in the absence of template occurs
primarily due to bad quality of Taq Polymerase. This often happens
when we change the source of Taq or if it has been handled by wrong
hands to contaminate it with some other template. Just a dip of
contaminated tip is enough to do the same. Always keep your tube of
taq for use by you only. However as suggested by 'Wo' run a parellel
amplification using new taq or taq from other source. Taq is produced
by recombinant clones. During its purification E.coli DNA is sometime
not removed completely and this DNA serves as template in NTRs. Adding
DNase and then inactivating is always problematic and is extremely
difficult to completely inactivate. All the best

On Mon, Sep 20, 2010 at 10:34 PM,  <methods-request from oat.bio.indiana.edu> wrote:
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> Today's Topics:
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>   1. Re: RE: non-specific bands in NTC (Cathal Garvey)
>   2. Re: non-specific bands in NTC (WS)
>   3. Re: non-specific bands in NTC (Cathal Garvey)
>   4. RE: RE: non-specific bands in NTC (Dunowska, Magda)
>   5. RE: non-specific bands in NTC (Dunowska, Magda)
>   6. Re: non-specific bands in NTC (Duncan Clark)
>   7. Re: non-specific bands in NTC (DK)
>
>
> ----------------------------------------------------------------------
>
> Message: 1
> Date: Sun, 19 Sep 2010 10:40:29 +0100
> From: Cathal Garvey <cathalgarvey from gmail.com>
> Subject: Re: RE: non-specific bands in NTC
> To: "Dunowska, Magda" <M.Dunowska from massey.ac.nz>
> Cc: "methods from magpie.bio.indiana.edu" <methods from magpie.bio.indiana.edu>,
>        Peter Ellis <pjie2 from cam.ac.uk>
> Message-ID:
>        <AANLkTi=y-KkW3MHvWkXfU7Wv4i_KWbxfx_KzAg4Z=rKF from mail.gmail.com>
> Content-Type: text/plain; charset=ISO-8859-1
>
> Hi Magda,
> It is possible that you are seeing amplification of the trace expression
> plasmid from production of the polymerase enzyme. To rule it out I guess
> you'd have to sequence a large band and blastn the result?
>
> ---
> Twitter: @onetruecathal
> Sent from my beloved Android phone.
>
> On 19 Sep 2010 02:57, "Dunowska, Magda" <M.Dunowska from massey.ac.nz> wrote:
>
> Just to clarify my previous email: NTC is "no template control" in the PCR
> run. It contains no template, so it is a type of a negative control. The
> difference between NTC tube and other negative tubes is that NTC has no
> template nucleic acids at all, while other negative samples have template
> nucleic acids in them, they are just negative for a specific sequence that I
> am looking for - I am looking for viral sequences in animal tissues. As I
> specified in my original email, the NTC tubes contain exactely the same
> master mix as all the other tubes - this is why I am so puzzled by what I
> see on the gel...Sorry again for not defining the abbreviation - Magda
>
> ________________________________________
> From: methods-bounces from oat.bio.indiana.edu [methods-bounces from ...
>
> On 17/09/2010 15:09, Nick Theodorakis wrote:
>> On Sep 17, 7:59 am, WS<novalidaddr... from nurfuerspam.de>...
>
>
> ------------------------------
>
> Message: 2
> Date: Sun, 19 Sep 2010 02:31:26 -0700 (PDT)
> From: WS <novalidaddress from nurfuerspam.de>
> Subject: Re: non-specific bands in NTC
> To: methods from net.bio.net
> Message-ID:
>        <93e77d22-8a77-45da-8cf3-89fce84a65be from k30g2000vbn.googlegroups.com>
> Content-Type: text/plain; charset=ISO-8859-1
>
> Dear Magda,
>
> might be that your polymerase contains traces of DNA from the host it
> was produced in. To confirm, add some DNAse to an aliquot of your
> mastermix, incubate say 1hr at 37 degC (or overnight if you like) and
> then kill the DNAse by heating to 95degC for say 10 minutes (longe is
> not so critical, but polymerase of course will decrease in activity).
> Then redo your assay. Include a control that demonstrates that DNAse
> is really gone; a control plasmid in a reasonable concentration should
> be amplified.
>
> Alternatively/additinally, you might try to switch polymerase batch/
> brand/type, too.
>
> Hope that helps,
>
> Wo
>
>
> ------------------------------
>
> Message: 3
> Date: Sun, 19 Sep 2010 19:39:26 +0100
> From: Cathal Garvey <cathalgarvey from gmail.com>
> Subject: Re: non-specific bands in NTC
> To: WS <novalidaddress from nurfuerspam.de>
> Cc: methods from magpie.bio.indiana.edu
> Message-ID:
>        <AANLkTimeaRiCYgd6Srm2n3pHzuCK4m9Yu5fFSx+kMDoP from mail.gmail.com>
> Content-Type: text/plain; charset=ISO-8859-1
>
> REmember to add primers after DNAse.. seems obvious but I make stupid
> mistakes daily.
>
> ---
> Twitter: @onetruecathal
> Sent from my beloved Android phone.
>
> On 19 Sep 2010 18:17, "WS" <novalidaddress from nurfuerspam.de> wrote:
>
> Dear Magda,
>
> might be that your polymerase contains traces of DNA from the host it
> was produced in. To confirm, add some DNAse to an aliquot of your
> mastermix, incubate say 1hr at 37 degC (or overnight if you like) and
> then kill the DNAse by heating to 95degC for say 10 minutes (longe is
> not so critical, but polymerase of course will decrease in activity).
> Then redo your assay. Include a control that demonstrates that DNAse
> is really gone; a control plasmid in a reasonable concentration should
> be amplified.
>
> Alternatively/additinally, you might try to switch polymerase batch/
> brand/type, too.
>
> Hope that helps,
>
> Wo
>
> _______________________________________________
> Methods mailing list
> Methods from net.bio.net
> http://www....
>
>
> ------------------------------
>
> Message: 4
> Date: Mon, 20 Sep 2010 08:43:31 +1200
> From: "Dunowska, Magda" <M.Dunowska from massey.ac.nz>
> Subject: RE: RE: non-specific bands in NTC
> To: Cathal Garvey <cathalgarvey from gmail.com>
> Cc: Peter, "methods from magpie.bio.indiana.edu"
>        <methods from magpie.bio.indiana.edu>,       Ellis <pjie2 from cam.ac.uk>
> Message-ID:
>        <92FDFD8B26EB6542B1E1BF017BB998D1638A75F637 from TUR-EXCHMBX.massey.ac.nz>
> Content-Type: text/plain; charset="us-ascii"
>
> This is a good thought! Thank you - trace amounts of the expression plasmid would not have crossed my mind. I am not sure whether I'd have enough DNA to cut out of a gel/clone/sequence (the bands are very weak), but I can certainly try...Magda
>
> From: Cathal Garvey [mailto:cathalgarvey from gmail.com]
> Sent: Sunday, 19 September 2010 9:40 p.m.
> To: Dunowska, Magda
> Cc: methods from magpie.bio.indiana.edu; Peter Ellis
> Subject: Re: RE: non-specific bands in NTC
>
>
> Hi Magda,
> It is possible that you are seeing amplification of the trace expression plasmid from production of the polymerase enzyme. To rule it out I guess you'd have to sequence a large band and blastn the result?
>
> ---
> Twitter: @onetruecathal
> Sent from my beloved Android phone.
> On 19 Sep 2010 02:57, "Dunowska, Magda" <M.Dunowska from massey.ac.nz<mailto:M.Dunowska from massey.ac.nz>> wrote:
>
> Just to clarify my previous email: NTC is "no template control" in the PCR run. It contains no template, so it is a type of a negative control. The difference between NTC tube and other negative tubes is that NTC has no template nucleic acids at all, while other negative samples have template nucleic acids in them, they are just negative for a specific sequence that I am looking for - I am looking for viral sequences in animal tissues. As I specified in my original email, the NTC tubes contain exactely the same master mix as all the other tubes - this is why I am so puzzled by what I see on the gel...Sorry again for not defining the abbreviation - Magda
>
> ________________________________________
> From: methods-bounces from oat.bio.indiana.edu<mailto:methods-bounces from oat.bio.indiana.edu> [methods-bounces from ...
>
> On 17/09/2010 15:09, Nick Theodorakis wrote:
>> On Sep 17, 7:59 am, WS<novalidaddr... from nurfuerspam.de<mailto:novalidaddr... from nurfuerspam.de>>...
>
>
> ------------------------------
>
> Message: 5
> Date: Mon, 20 Sep 2010 08:45:23 +1200
> From: "Dunowska, Magda" <M.Dunowska from massey.ac.nz>
> Subject: RE: non-specific bands in NTC
> To: Cathal Garvey <cathalgarvey from gmail.com>, WS
>        <novalidaddress from nurfuerspam.de>
> Cc: "methods from magpie.bio.indiana.edu" <methods from magpie.bio.indiana.edu>
> Message-ID:
>        <92FDFD8B26EB6542B1E1BF017BB998D1638A75F641 from TUR-EXCHMBX.massey.ac.nz>
> Content-Type: text/plain; charset="us-ascii"
>
> Thanks! I will try this and see what happens - Magda
>
>
> -----Original Message-----
> From: methods-bounces from oat.bio.indiana.edu [mailto:methods-bounces from oat.bio.indiana.edu] On Behalf Of Cathal Garvey
> Sent: Monday, 20 September 2010 6:39 a.m.
> To: WS
> Cc: methods from magpie.bio.indiana.edu
> Subject: Re: non-specific bands in NTC
>
> REmember to add primers after DNAse.. seems obvious but I make stupid
> mistakes daily.
>
> ---
> Twitter: @onetruecathal
> Sent from my beloved Android phone.
>
> On 19 Sep 2010 18:17, "WS" <novalidaddress from nurfuerspam.de> wrote:
>
> Dear Magda,
>
> might be that your polymerase contains traces of DNA from the host it
> was produced in. To confirm, add some DNAse to an aliquot of your
> mastermix, incubate say 1hr at 37 degC (or overnight if you like) and
> then kill the DNAse by heating to 95degC for say 10 minutes (longe is
> not so critical, but polymerase of course will decrease in activity).
> Then redo your assay. Include a control that demonstrates that DNAse
> is really gone; a control plasmid in a reasonable concentration should
> be amplified.
>
> Alternatively/additinally, you might try to switch polymerase batch/
> brand/type, too.
>
> Hope that helps,
>
> Wo
>
> _______________________________________________
> Methods mailing list
> Methods from net.bio.net
> http://www....
> _______________________________________________
> Methods mailing list
> Methods from net.bio.net
> http://www.bio.net/biomail/listinfo/methods
>
>
>
> ------------------------------
>
> Message: 6
> Date: Mon, 20 Sep 2010 10:11:47 +0100
> From: Duncan Clark <blackhole from abuse.plus.com>
> Subject: Re: non-specific bands in NTC
> To: methods from net.bio.net
> Message-ID: <YUGLMrDTVylMFAQr from abuse.plus.com>
> Content-Type: text/plain;charset=us-ascii;format=flowed
>
> Historians believe that in newspost
> <mailman.185.1284934038.1545.methods from net.bio.net> on Mon, 20 Sep 2010,
> "Dunowska, Magda" <M.Dunowska from massey.ac.nz> penned the following
> literary masterpiece:
>>Thanks! I will try this and see what happens - Magda
>
> And if your pancreatic DNAse is not pure enough it will contain a
> protease that nicely cuts up Taq polymerase :-(
>
> Duncan
> --
> I love deadlines. I especially like the whooshing noise they make as
> they go flying by.
>
> Duncan Clark
> GeneSys Ltd.
>
>
> ------------------------------
>
> Message: 7
> Date: Mon, 20 Sep 2010 14:05:49 GMT
> From: dk from no.email.thankstospam.net (DK)
> Subject: Re: non-specific bands in NTC
> To: methods from net.bio.net
> Message-ID: <5TJlo.47157$0O3.43245 from newsfe11.iad>
>
> In article <YUGLMrDTVylMFAQr from abuse.plus.com>, Duncan Clark <news from genesysltd.co.uk> wrote:
>>Historians believe that in newspost
>><mailman.185.1284934038.1545.methods from net.bio.net> on Mon, 20 Sep 2010,
>>"Dunowska, Magda" <M.Dunowska from massey.ac.nz> penned the following
>>literary masterpiece:
>>>Thanks! I will try this and see what happens - Magda
>>
>>And if your pancreatic DNAse is not pure enough it will contain a
>>protease that nicely cuts up Taq polymerase :-(
>
> Add 1 mM of PMSF (AEBSF if the final concentration of ispropanol
> < 1% is a problem) into DNAse stock and the problem is solved.
>
> DK
>
>
> ------------------------------
>
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>
> End of Methods Digest, Vol 64, Issue 5
> **************************************
>



-- 
Dr V K Gupta
Sr Microbiologist (Mol Biology)
IMBL, Department of Entomology
Pun. Agric. Univ., Ludhiana (Pb)-141004- India
M: 081465-55515



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