(none)

Bruce Roe broe at aardvark.ucs.uoknor.edu
Sat Jun 6 08:43:00 EST 1992


In article <9206050920.AA03703 at genbank.bio.net>, URA1335 at FRCITI51.BITNET writes...
> 
>                Hi! Netters,
> 
>        I have a PCR problem!
>        According to Tm=2AT+4CG, we calculated a Tm=66
>        for oligo 1, and tm=58 for oligo 2.
> 
>        Here is my question : how come that i had still
>        PCR bands ( that I have no any idea about their
>        specificity ) when i did my PCR reaction
>        at an annealing temperature = 60 degrees ????
>        just 2 degrees above Tm.
> 
>        is it necessary to continue to do PCR in highter
>        temperature above Tm ????.
> 
>        Do you know any published references about that ????
> 
>                                        Dr ARAL Serdar
> 
>                                EARN::"URA1335 at FRCITI51.BITNET"

Isn't the Tm defined as the temperature where 50% is duplex and
50% is not?  Think about it?  Even if only a few % of the primers
bind and at only 2 degrees above the Tm you probably have 40% bound.
Once the polymerase extends, the Tm for these longer pieces will
increase and thus you'll get the amplification you observed.

As for a general note regarding Tm calculations, the formula you
are using is a "crude estimate" of the Tm and thus not very exact.
Check out a Physical Biochemistry Text such as Cantor and Schimmel.
There also is an IRL press paperback on Hybridization that is pretty
decent and may give you additional info.

Cheers...........bruce
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