Polymerase Chain Reactions

How to choose a DNA Primer

Open the link above to find the homepage for the NCBI website. From the search bar, select "Gene" in the drop down menu and search for the strain you would like.



You can filter the Search Results by "CCDS", which includes sequence databases we will need to access the sequences. Select the gene you desire and scroll down until you can find the CCDS link, it should be under "Related Information" on the right side of the page.




In the CCDS database, you may be presented with several options for CCDS IDs, choose one and select "C" in the Links column. Towards the bottom of the page, you will see the nucleotide sequence. Copy the sequence for future use in Benchling, which we will use to create the primer we would like.

Using Benchling to create a primer

Open Benchling and create an account if you do not already have one. Click the plus icon on the left-side toolbar and select "DNA Sequence", this will allow you to insert the DNA sequence of your choice. Paste the sequence from above in to the blank space on the screen, it will look like this after it has been pasted. The nucleotides highlighted in pink are the ones that I chose for the forward primer.



To create the primer, select the forward/reverse arrows icon on the right-side toolbar, and complete the file title and choose the correct topology for your gene. You can also choose to create a single primer or primer pair using the drop-down menu on the top left of the page. For our purposes, we will be using the Primer Pair option. Go back to the "Sequence map option", and select the set of genes you would like. In "Design Primer", choose "Set from Selection" and choose "Forward". Repeat this process for the "Reverse: selection. From here, you have several options to design your primer. You want the Tm to be to greater than 10 degrees Celcius apart from one another or it will not yield accurate results in the PCR. Name the primers and save them for ordering in the future.