non-specific bands in NTC

Dunowska, Magda via methods%40net.bio.net (by M.Dunowska from massey.ac.nz)
Sat Sep 18 17:34:11 EST 2010


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 oat.bio.indiana.edu] On Behalf Of Peter Ellis [pjie2 from cam.ac.uk]
Sent: Saturday, September 18, 2010 6:43 AM
To: methods from magpie.bio.indiana.edu
Subject: Re: non-specific bands in NTC

On 17/09/2010 15:09, Nick Theodorakis wrote:
> On Sep 17, 7:59 am, WS<novalidaddr... from nurfuerspam.de>  wrote:
>> NTC tubes? WTH is that? Neg Temp Coeff?
>>
>> /Wo
>
> I would have guessed "no template control" except that I'm not sure
> how that would be different than her negative samples which she says
> are clean.

Depends what you're testing for - a "negative" sample could be a
template which won't amplify.

e.g. genotyping for the presence/absence of a transgene.  Positive would
be DNA from a known transgenic animal, negative would be DNA from a
known wildtype animal, no template control would have no template.  In
that case the negative control would be controlling for the specificity
of the PCR in detecting the tg against genomic background, while the no
template control would be testing for contamination in one or more of
the reagents.

Magda: What size are the erroneous products?  Some sort of primer dimer
is certainly a possibility.

Peter
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