Hi,

It depends on many conditions, but some main tips would be considered:

1. The length of primers: it is better to be 18-24 nucleotides (the forward and reverse primers ideally have the same length).

2. The annealing temperature (it's related to 1).

3. The melting temperature (Tm): primer pairs should have Tms within 5°C of each other.

4. The GC content (Ideal GC content is 40-60%).

5. The length of the PCR product that is related to the forward and reverse primers binding sites.

6. Be aware of Taq DNA polymerase limitation: standard PCR using Taq DNA polymerase is generally limited to amplifications up to 4 kb.

7. Using the proper buffer for primer dilution: TE buffer or nuclease-free water can be used.

Besides, use high-quality, purified DNA templates to have good results.

Good luck

A classic article on PCR primer design is attached (download from the link), go through it first carefully. Subsequently, you can also use the online tools available freely (link below).

best wishes,

https://genome.cshlp.org/content/3/3/S30.full.pdf

https://www.thermofisher.com/in/en/home/life-science/oligonucleotides-primers-probes-genes/custom-dna-oligos/oligo-design-tools.html?gclid=CjwKCAjwgqejBhBAEiwAuWHioBHUni-Ka5PfWER4qsuDy6D8OZYL3SxnTq1TvrsyMaAyS7eLZcfashoCrysQAvD_BwE&s_kwcid=AL!3652!3!506722412555!p!!g!!how%20to%20design%20a%20primer&ef_id=CjwKCAjwgqejBhBAEiwAuWHioBHUni-Ka5PfWER4qsuDy6D8OZYL3SxnTq1TvrsyMaAyS7eLZcfashoCrysQAvD_BwE:G:s&s_kwcid=AL!3652!3!506722412555!p!!g!!how%20to%20design%20a%20primer!12555269440!117284659657&cid=bid_mol_pch_r01_co_cp1358_pjt0000_bid00000_0se_gaw_nt_pur_awa&gad=1

Hi Aleena

Blast is not a primer development tool, but it can be used to assess primer compatibility with the target sequence. The primer sequence should ideally be conserved. By using Blast, you can obtain the top 100 sequences related to your sequence. The higher the coverage and identity (100%) among the top 100 sequences, the better the primer's quality.

@Aydin Hassanzadeh@Ahemed Elsadek@Sibnarayanan Datta thank you so much for your valuable suggestions

Hello, there are some free software that can be used to analyze the quality of the selected primers. I am using GeneRunner software to check if the primers have hairpins, loops, primer dimers etc. Here is a step by step process to assess. Hope you find it useful. https://www.youtube.com/watch?v=eTz1X5hGQp4