What are secondary structures in primers?

Primer Secondary Structures: Presence of the primer secondary structures produced by intermolecular or intramolecular interactions can lead to poor or no yield of the product. They adversely affect primer template annealing and thus the amplification. They greatly reduce the availability of primers to the reaction.

How do you reduce the secondary structure of a primer?

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

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

How do you determine DNA template for PCR?

The total number of copies of double stranded DNA may be calculated using the following equation: Number of copies of DNA = (DNA amount (ng) x 6.022×1023) / (length of DNA x 1×109 ng/ml x 650 Daltons) Calculating the number of copies of DNA is used to determine how much template is needed per reaction.

What is secondary structure in PCR?

When designing primers for PCR, two types of secondary structures should be analyzed: dimers and hairpins. For dimers, both self- and hetero-dimers should be reviewed. In general, the ΔG value for dimer analysis should be between 0 to −9 kcal/mole for optimal design.

Is a primer dimer a secondary structure?

When designing primers for PCR, two types of secondary structures should be analyzed: dimers and hairpins. For dimers, both self- and hetero-dimers should be reviewed.

Why internal secondary structures are not preferred for primers?

Why internal secondary structures are not preferred for primers? Explanation: Internal structures are not preferred because if they are present the primer may fold back on itself. As the primer folds back on it, it is not available for the template.

What is the best DNA concentration for PCR?

The concentration of each primer should be between 0.1 and 0.5 µM. For most applications 0.2 µM produces satisfactory results. Too high primer concentrations increase the chance of mispriming, which results in nonspecific PCR products.

What is a good DNA concentration for PCR?

Template DNA Nevertheless, the composition or complexity of the DNA contributes to optimal input amounts for PCR amplification. For example, 0.1–1 ng of plasmid DNA is sufficient, while 5–50 ng of gDNA may be required as a starting amount in a 50 µL PCR.