What is a good Tm for primers?

What is a good Tm for primers?

Primer melting temperature (Tm) by definition is the temperature at which one half of the DNA duplex will dissociate to become single stranded and indicates the duplex stability. Primers with melting temperatures in the range of 52-58°C generally produce the best results.

How do you calculate annealing temperature of Tm primers?

The optimal annealing temperature (Ta Opt) for a given primer pair on a particular target can be calculated as follows: Ta Opt = 0.3 x (Tm of primer) + 0.7 x (Tm of product) – 14.9; where Tm of primer is the melting temperature of the less stable primer-template pair, and Tm of product is the melting temperature of the …

How do you use the NEB TM on a calculator?


  1. Select the product group of the polymerase or kit you plan to use.
  2. Select the polymerase or kit from the list of products.
  3. If needed, modify the recommended primer concentration.
  4. Enter primer sequences (with up to 3 ambiguous bases). Spaces allowed.

How high can primer Tm be?

As Mathis Wolter said the ideal TM of the primers must be between 50-60 C. Your TM is too high try to lower it down to at least 65-60 C, you can use temps of -5 C or + 5 C based on your Primers TM (i.e if you have a TM of 60C you can go either 55 C or 65 C).

What is a good GC content for primers?

Aim for the GC content to be between 40 and 60% with the 3′ of a primer ending in G or C to promote binding. This is known as a GC Clamp. The G and C bases have stronger hydrogen bonding and help with the stability of the primer.

Is TM annealing temperature?

And the annealing temperature is that temperature where primers successfully bind. Therefore the Annealing temperature should be less than the Tm of primers. Usually annealing temperature is 55-60˚C, but if we lower the temperature i.e. 45-55˚C it promotes binding to the DNA.

Why TM value is important in PCR?

For PCR, the melting temp plays an important role for avoiding unspecific binding to DNA sequences. It controls/allows binding to specific target sequences of the DNA in the “annealing” step of PCR. Tm of primers ist – among other things – dependent on the GC content of the oligo sequence.

How will Tm change as the primer length increases?

As a rule of thumb, the Tm of the primers can be estimated by adding 2°C for each A or T and 4°C for each G or C. The Tm of a nucleic-acid duplex increases both with its length and with increasing GC content. Make sure the Tm difference of your custom primers is no more than 5°C between the pair.

What happens if primer melting temperature is too high?

Primer Annealing Temperature: The primer melting temperature is the estimate of the DNA-DNA hybrid stability and critical in determining the annealing temperature. Too high Ta will produce insufficient primer-template hybridization resulting in low PCR product yield.

Why is it recommended to have a 40% 60% GC content?

What is Ta and Tm of the primer?

What are the factors affecting TM?

The melting temperature depends on a variety of factors, such as the length of DNA [11], [12] (shorter pieces tend to melt more easily, [13]), the nucleotide sequence composition [14]–[16], salt concentration (ionic strength of the added salt) [14]–[15], [17] and generally lies between 50°C and 100°C.

What is TM on a denaturation curve?

The Temperature of Melting (Tm) is defined as the temperature at which 50% of double stranded DNA is changed to single-standard DNA. The higher the melting temperature the greater the guanine-cytosine (GC) content of the DNA.