5 replies to this topic

[travelhappily]

why are primer dimers produced ?
I use these primers  before in PCR.
But in PCR screening,they can't be used. Primer dimers are formed.

[Ahrenhase]

travelhappily, on May 13 2009, 12:23 PM, said:

why are primer dimers produced ?
I use these primers  before in PCR.
But in PCR screening,they can't be used. Primer dimers are formed.

Primer dimers are formed when there is complementarity among the bases that comprise the primers, or something like that.  You know chargaff's rule, A:T and C:G, well this can happen b.w the primers too.  Primer dimers are never good, but if their strenght level is under -4 kCal/mol I wouldn't worry about them, atleast that's what I was told.

[travelhappily]

Ahrenhase, on May 13 2009, 01:36 PM, said:

Primer dimers are formed when there is complementarity among the bases that comprise the primers, or something like that.  You know chargaff's rule, A:T and C:G, well this can happen b.w the primers too.  Primer dimers are never good, but if their strenght level is under -4 kCal/mol I wouldn't worry about them, atleast that's what I was told.

Some one explained to me that  primer dimers always exist since there is a competence between primer itself and template.So that means primer 1 will base pair with itself and primer 2 will base pair with itself too.Not between 1 and 2.

I have no problem in this pair of primers since I have used them successfully before several times.There shouldn't be some complementary problem in primer 1 and 2.

[mastermi]

travelhappily, on May 24 2009, 07:14 PM, said:

Some one explained to me that  primer dimers always exist since there is a competence between primer itself and template.So that means primer 1 will base pair with itself and primer 2 will base pair with itself too.Not between 1 and 2.

I have no problem in this pair of primers since I have used them successfully before several times.There shouldn't be some complementary problem in primer 1 and 2.

???But if you don't have inverted repeats in your primer sequence you won't have complementarity either???

[warsel]

travelhappily, on May 24 2009, 10:14 AM, said:

Some one explained to me that  primer dimers always exist since there is a competence between primer itself and template.So that means primer 1 will base pair with itself and primer 2 will base pair with itself too.Not between 1 and 2.

This sounds to me like someone was wrong.
Properly designed primers do not, in general, self-dimerize at the temperatures that PCRs are run at.

They definitely do not compete with the template unless designed in such a way (as in some non-realtime qauantitative PCR protocols where you use competition to quantify your product).

[lsek]

Primer dimer around deltaG -9kcal/mole is still acceptable, though higher is still the best.

Now, by saying PCR screening, do you mean colony screening? Did your non-template control resulted in primer dimer as well? If NTC don't have that, then it must be contaminants from lysate that causes the "primer dimer". If NTC has too, surely it's primer dimer.

Edited by lsek, 29 May 2009 - 07:39 AM.