Let’s start with designing detection primers, helpful in knowing if a sequence is present in a larger context like a plasmid, and reliably amplifies on a gel. Start by obtaining the sequence data of your desired amplicon and up to 10,000bp of the genetic context that amplicon target resides in. This can be a fully sequenced plasmid or a chunk of chromosomal DNA. Anything larger will require local installation of Primer3 and a linux machine to generate. For this example, we’ll use my pIDMv5K-GREEN plasmid as reference. Feel free to download the sequence file and follow along. File is in SnapGene format which has a free Viewer program you can download here.
Open a web browser and open Primer3plus.com. You will be greeted with the main interface of the program.
Click on the General Settings tab and let’s change a few parameters. For this tutorial let’s first try to make some detection primers. Detection primers are ideal primers found inside your gene of interest that produce an amplicon of about 500bp, where Primer3 selects the best primer pair within that sequence to get the job done. These primers test for the presence of a gene and, in my experience over the last decade, have never failed to produce bands during PCR. The first think I do is change the Product Size Ranges from the long default list to a simple 450-550. Note this MUST be a range and not a single value. If you want a precise size, just give it a range of size-1 to size desired (e.g. 99-100 for a 100bp amplicon). Next we go to the Primer Size fields where I like having an optimal oligo size (Opt) to be 25bp. For the Primer Tm I try to ALWAYS design my primers to have a Tm of 60C so I change the Opt field of Primer Tm to 60.
Now we can shift back to the Main tab, where we then copy and paste the entire plasmid you wish to amplify from. If your plasmid is larger than the max input size of 10,000bp, select a 10kbp chunk where your gene of interest is in the middle of that chunk. We want to include the entire genetic context of your target amplicon so that Primer3 can screen for secondary amplifications. If you only plug in your gene of interest there could be a risk of one of your primers binding elsewhere.
Now go back to your plasmid editor and copy the sequence of your gene of interest. I’m using SnapGene as my plasmid viewer and Microsoft Edge as my web browser. While in the Primer3 input box, press Ctrl + F or the search command in your browser and plug in your copied gene. This will highlight that sequence in the textbox.
Drag your cursor over the highlighted region to highlight it again, and then click the “{ Included }” button. This will suddenly generate some numbers in the Included Region textbox below, marking the start and stop position of the region to include in the primer search. This is a command known to Primer3 which flags the bracketed sequence as an Included Region. Primer3 will look for ideal primer sequences only inside this region, but will take the outside sequences as reference for secondary amplification.
Now that we have our parameters, template sequence, and the region we wish to design primers for, we can click the green Pick Primers button on the top right. This will generate primers, where the top two primers are the best-scoring ones given myriad thermodynamic and contextual criteria.
Copy those two primers into your plasmid viewer or plasmid design tool and double check if they bind where you want them. In SnapGene, you can add primers to a plasmid file via the Ctrl + R shortcut in Windows (likely Command + R or Option + R in Mac). The primers are all 5’ to 3’ so they are ready to be copied and pasted into an order form of your favorite oligo synthesis company. The reverse primer is already in the reverse compliment so no need to reformat the sequence. In this example the two primers generated are:
