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.

Which GC content of primers is least desirable for a PCR?

Primers with 40% to 60% GC content ensure stable binding of primer and template. However, sequences containing more than three repeats of sequences of G or C in sequence should be avoided in the first five bases from the 3′ end of the primer because of the higher probability of primer-dimer formation.

What happens if annealing temp is too low?

If the annealing temperature is too low, primers may bind nonspecifically to the template. The rule of thumb is to use an annealing temperature that is 5°C lower than the Tm of the primer.

How do you prevent primer dimers?

i suggest one (or more) of the following solutions:

increase the annealing temperature.
increase time\ temperature of template denaturation.
decrease primers concentration(10 pmol will be OK)
use a PCR enhancer such as DMSO.
Check out your template.
use high quality Tag.

What is a primer dimer how do they form?

A primer dimer (PD) is a potential by-product in the polymerase chain reaction (PCR), a common biotechnological method. As its name implies, a PD consists of two primer molecules that have attached (hybridized) to each other because of strings of complementary bases in the primers.

What happens if primers are too short?

Short primers produce inaccurate, nonspecific DNA amplification product, and long primers result in a slower hybridizing rate.

How do I test my primer dimer?

Use the “”Hetero-Dimer”” button in the OligoAnaylzer® program to test for primer dimers. Enter the sequence of your forward primer into the sequence box, and then click ‘Hetero-Dimer. ‘ This will open a second box below the original sequence box, in which you enter the sequence of your reverse primer.