Inosine residues and effect on Tm

Marianna Max drmax at casbah.acns.nwu.edu
Fri Jul 2 21:00:25 EST 1993


In article <21183g$pi0 at nuscc.nus.sg> bchtantw at nuscc.nus.sg (Dr Tan Tin Wee) writes:
>
>Does anyone know how I can calculate the Tm of an oligonucleotide
>containing inosine residues?
>
>Any help will be gratefully received.
>I will summarise if there is a demand.
>
>Thanks in advance.
>
>Tin Wee.
>
>--
>    Tin-Wee TAN, PhD.                  /  INTERNET: bchtantw at nuscc.nus.sg
>    Dept of Biochemistry               /  BITNET:   BCHTANTW at NUSVM
>    National University of Singapore   /  Tel:      (65)-772-3678
>    Singapore 0511                     /  Fax:      (65)-779-1453


here's the equation I use 

60 + .41 x %GC content - 600/length of the primer 

and I only count a positon if all possible nucleotides at that position
are either a G or a C. 

I run my PCR 5 degrees below the Tm. I also calculate with and without any
restriction sites that might be added onto the primer and run several
cycles to allow annealing  without the restriction site followed by 30 or so
cycles appropriate for annealing with the restriction site. 

Depends on what you are trying to do of course.

Max



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