I've been reading about the length of the primers and I found that is around 18-30 bases and the shorter the primers are, the more efficiently they will bind or anneal to the target. However, I added like 8 bp to mine and they became more stable, it increased the concentration and the Tm changed. Please help me to understand what happend because I haven't found information about that, and if you have a paper or link to support your answer, that would be great.
Thanks!
Dear Isabel Cristina Hernandez Jiron
Congrats on your success in designing a stable primer!
Actually primer designing is crucial and pivotal! There are actually some unique factors to declare the best primer for a particular sequence. Adjusted Tm value, GC content (40%-60%, 50% is more preferable), product length (100 -800), primer sequence length ( 20-25), and the hairpin loop can be considerable. But it depends on the way of your research.
Dear Isabel, scientifically speaking there is a rule of thumb that longer primers (i.e; above 18 bp) are more specific if you maintain a good GC%. Primer specificity comes with its Ta (annealing temperature), which is directly primer length dependent. A best Ta value achieves more specificity for a primer pair and in long primers the Ta is more. This high Ta of primer avoids mis-extensions of incorrect bp at 3' end and if there are any secondary structures present on our template DNA, so due to these long primers this won't affect much for our product amplicon generation. Also as a reminder, if your GOI or target is more than 10 kbp so always design primer at least 25 - 35 mer long.
Regards...
A good length for PCR primers is generally around 18-30 bases, so that Specificity of primers usually are dependent on length and annealing temperature.
Therefore, high length of primer contain a higher number of G-C base pairs will be more strongly bonded together, more stable, and will have a higher melting temperature.
Dear Isabel Cristina Hernandez Jiron
Short primer=non specific binding
Long primer= Specific binding.
However if you took too long primer, then you may not find a good homology reason in the target site. So ideally primer should be
Length= 20-24bp
GC=40-60%
Ends=Primer should have G or C at the ends so they can not prone to remove. But remember should not put G on one end and C on other end or vise-versa. Otherwise primers may form loop and you may not detect any PCR amplicon
Primer Stability= should be in positive
GC clamp= Primer should be free of GC clamp
You may use Clone Manger 9.0 or other version for better experience
I hope this will help you.
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