Can I use this rule of Melting Temperature = 4(G+C) +2(A+T) “Wallace rule
or there is a better formula to calculate Tm. I am using this buffer
10X ViBuffer A (without MgCl2} 500mM KCl, 100mM Tris-HCI (PH 9.1 at 20°C) and 0.1 % Triton™ X-100. The buffer is optimized for use with 0.1-0.2mM of each dNTP. in my PCR. Any help is really appreciated. Thank you
Primers' Tm calculation will never be exact, whatever bioinformatic tool you will use, since it depends on too many factors that you will not be able to control (monovalent salt concentration, divalent salt concentration, any additives in your reaction such as the glycerol from the Taq, DMSO if you will happen to need it, etc., and even the primer concentration you will end up using due to the competition with their intramolecular secondary structures or intermolecular interactions)... therefore I would not be too picky with finding exact numbers. For primers up to 18 nt in length the Wallace rule works very well, however rough it is. For longer primers you might want to use a bioinformatic tool, or rely on what the company datasheet tells you.
I would rather suggest paying attention at the 3' side of your primers, when you design them, which has a lot more importance, since it's kinetic stability is fundamental for the efficiency of the Taq polymerase starting the amplification. For example your F primer ends with TTGGG, a very strong and stable sequence that will require higher annealing temp. to avoid unspecific binding of your primer due to the 3 consecutive last Gs. Vice versa, your F primer ends with GATTG, a much weaker sequence due to the 3 consecutive A/T pairings and only a last G, which will not allow you to raise much the annealing temp. unless your PCR will lose efficiency. In practice your primers appear to have a contradictory behaviour, at least in theory. Keep in mind practice can be very different, since the kinetics are too complex to be able to predict everything. Anyway, it is a good rule to try and design primers with similar theoretical thermodynamic and kinetic properties. In general, and mostly if the GC content of your primers is below 50%, as observed, it helps designing primers which contain in the last 5 3' nts at least 3 G/C pairings, with the last two nts being a GG or CC couple (never use GC because it can give rise to primer dimers) and space the other G or C with 1 or 2 A/Ts.
It also helps blasting you last 7 nts on the 3' side against the sequence of your gene (sense and antisense)... if the sequence finds more than 1 or 2 unspecific matches I would change the primer, it's not a good sign of specificity... imagine how many matches could it have on the whole transcriptome or genome...!
Obviously the Wallace rule is not accurate enough, it is only a rough way to estimate the tm of your oligos. By the way I always use a seqbuilder program for the preparation of my experiments, they have always a built-in software for oligo calculations. In addition to this I use to double check the parameters through online tools, indeed there are a lot of online tools useful for a lot of parameters such as selfdimer-heterodimer efficiency, GC content, TM in different salt concentrations, secondary structures/hairpins/stem-loops. I like so much this one http://eu.idtdna.com/calc/analyzer but you can find a lot more useful tool googling, for example http://www.basic.northwestern.edu/biotools/oligocalc.html.
In any case I'd like to suggest you to rise your oligos TM by increasing GC content, because they're under 50°C checking under standard parameters.
Use this online tool:
http://www.bioinformatics.org/sms2/pcr_primer_stats.html
Hope it will help you.
You might also consider Primer3 for primer selections and optimizations.
http://bioinfo.ut.ee/primer3-0.4.0/
Lots of options and works fine.
Primers' Tm calculation will never be exact, whatever bioinformatic tool you will use, since it depends on too many factors that you will not be able to control (monovalent salt concentration, divalent salt concentration, any additives in your reaction such as the glycerol from the Taq, DMSO if you will happen to need it, etc., and even the primer concentration you will end up using due to the competition with their intramolecular secondary structures or intermolecular interactions)... therefore I would not be too picky with finding exact numbers. For primers up to 18 nt in length the Wallace rule works very well, however rough it is. For longer primers you might want to use a bioinformatic tool, or rely on what the company datasheet tells you.
I would rather suggest paying attention at the 3' side of your primers, when you design them, which has a lot more importance, since it's kinetic stability is fundamental for the efficiency of the Taq polymerase starting the amplification. For example your F primer ends with TTGGG, a very strong and stable sequence that will require higher annealing temp. to avoid unspecific binding of your primer due to the 3 consecutive last Gs. Vice versa, your F primer ends with GATTG, a much weaker sequence due to the 3 consecutive A/T pairings and only a last G, which will not allow you to raise much the annealing temp. unless your PCR will lose efficiency. In practice your primers appear to have a contradictory behaviour, at least in theory. Keep in mind practice can be very different, since the kinetics are too complex to be able to predict everything. Anyway, it is a good rule to try and design primers with similar theoretical thermodynamic and kinetic properties. In general, and mostly if the GC content of your primers is below 50%, as observed, it helps designing primers which contain in the last 5 3' nts at least 3 G/C pairings, with the last two nts being a GG or CC couple (never use GC because it can give rise to primer dimers) and space the other G or C with 1 or 2 A/Ts.
It also helps blasting you last 7 nts on the 3' side against the sequence of your gene (sense and antisense)... if the sequence finds more than 1 or 2 unspecific matches I would change the primer, it's not a good sign of specificity... imagine how many matches could it have on the whole transcriptome or genome...!
Just one little addition. The only way to be sure about primers functionality and specificty is by testing them under different conditions/dilutions/annealing temperatures and also making more than one primers pair and test them in different combinations. In silico predictions are useful but the benchwork never lie!
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