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, <[email protected]> wrote: > Send Methods mailing list submissions to > [email protected] > > To subscribe or unsubscribe via the World Wide Web, visit > http://www.bio.net/biomail/listinfo/methods > or, via email, send a message with subject or body 'help' to > [email protected] > > You can reach the person managing the list at > [email protected] > > When replying, please edit your Subject line so it is more specific > than "Re: Contents of Methods digest..." > > > Today's Topics: > > 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 <[email protected]> > Subject: Re: RE: non-specific bands in NTC > To: "Dunowska, Magda" <[email protected]> > Cc: "[email protected]" <[email protected]>, > Peter Ellis <[email protected]> > Message-ID: > <[email protected]> > 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" <[email protected]> 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: [email protected] [methods-boun...@... > > On 17/09/2010 15:09, Nick Theodorakis wrote: >> On Sep 17, 7:59 am, WS<[email protected]>... > > > ------------------------------ > > Message: 2 > Date: Sun, 19 Sep 2010 02:31:26 -0700 (PDT) > From: WS <[email protected]> > Subject: Re: non-specific bands in NTC > To: [email protected] > Message-ID: > <93e77d22-8a77-45da-8cf3-89fce84a6...@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 <[email protected]> > Subject: Re: non-specific bands in NTC > To: WS <[email protected]> > Cc: [email protected] > Message-ID: > <[email protected]> > 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" <[email protected]> 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 > [email protected] > http://www.... > > > ------------------------------ > > Message: 4 > Date: Mon, 20 Sep 2010 08:43:31 +1200 > From: "Dunowska, Magda" <[email protected]> > Subject: RE: RE: non-specific bands in NTC > To: Cathal Garvey <[email protected]> > Cc: Peter, "[email protected]" > <[email protected]>, Ellis <[email protected]> > Message-ID: > <92fdfd8b26eb6542b1e1bf017bb998d1638a75f...@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:[email protected]] > Sent: Sunday, 19 September 2010 9:40 p.m. > To: Dunowska, Magda > Cc: [email protected]; 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" > <[email protected]<mailto:[email protected]>> 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: > [email protected]<mailto:[email protected]> > [methods-boun...@... > > On 17/09/2010 15:09, Nick Theodorakis wrote: >> On Sep 17, 7:59 am, >> WS<[email protected]<mailto:[email protected]>>... > > > ------------------------------ > > Message: 5 > Date: Mon, 20 Sep 2010 08:45:23 +1200 > From: "Dunowska, Magda" <[email protected]> > Subject: RE: non-specific bands in NTC > To: Cathal Garvey <[email protected]>, WS > <[email protected]> > Cc: "[email protected]" <[email protected]> > Message-ID: > <92fdfd8b26eb6542b1e1bf017bb998d1638a75f...@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: [email protected] > [mailto:[email protected]] On Behalf Of Cathal Garvey > Sent: Monday, 20 September 2010 6:39 a.m. > To: WS > Cc: [email protected] > 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" <[email protected]> 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 > [email protected] > http://www.... > _______________________________________________ > Methods mailing list > [email protected] > http://www.bio.net/biomail/listinfo/methods > > > > ------------------------------ > > Message: 6 > Date: Mon, 20 Sep 2010 10:11:47 +0100 > From: Duncan Clark <[email protected]> > Subject: Re: non-specific bands in NTC > To: [email protected] > Message-ID: <[email protected]> > Content-Type: text/plain;charset=us-ascii;format=flowed > > Historians believe that in newspost > <[email protected]> on Mon, 20 Sep 2010, > "Dunowska, Magda" <[email protected]> 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: [email protected] (DK) > Subject: Re: non-specific bands in NTC > To: [email protected] > Message-ID: <[email protected]> > > In article <[email protected]>, Duncan Clark > <[email protected]> wrote: >>Historians believe that in newspost >><[email protected]> on Mon, 20 Sep 2010, >>"Dunowska, Magda" <[email protected]> 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 > > > ------------------------------ > > _______________________________________________ > Methods mailing list > [email protected] > http://www.bio.net/biomail/listinfo/methods > > 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 _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
