Detailed Steps on Primer Design

I list here the steps on how to design a good working primer.

1.       Get the sequence of your gene  from published literature for example OsNAS3.

2.       Using the TIGR Blast/ NCBI Blast, check the whole sequence if it specifies the gene of interest. It is important that the gene sequence is 100% homologous to the plant where the sequence is derived and the most important is that the whole sequence specifies only one distinct gene.

3.       Put your sequence inside the box of   Primer 3 online software. Specify in the software what are the parameters of the primer you want to have. Click generate. You would get something like this.

Important Notes:

When designing primers take note of the following:

GC content, melting temp (Tm) of primer, 3’ ends of the primer

PCR product size- It depends on what your purpose is. If your going to used the conventional PCR you can create a primer with a product size of 400bp and up. If you’re going to use the primer for real time PCR design a primers with 100-350 size only.  

4.       To be extra sure if your primer is really  specific,  Blast them again with NCBI blast. This would look like this. With the use of Vector NTI/Bioedit you can check the size of your PCR product.

5.       You are now ready to order your primers at SBS and proligo.