What are the steps involved in getting the reverse primer sequence?

Designing the reverse primer can be a bit challenging for two reasons- the way we communicate DNA sequences and the need to work with the template strand for primer design. Using software such as APE (A Plasmid Editor) can make designing the reverse primer much easier. Following are the steps to obtain the reverse primer sequence:

Step 1- Begin designing the reverse primer from the end of the sequence you have added to APE. Decide whether to include the stop codon in the primer based on your downstream workflow. If you want to add a tag or peptide to the C-terminal of the protein, remove the stop codon from the reverse primer to allow for proper translation. On the other hand, if you want to sub-clone the gene or express the protein without tags at the C-terminal, include the stop codon to prevent translational fusion of vector sequences. 

Step 2 - Select the sequence until the parameters such as the GC content and Tm (melting point) match the desired values. Keeping the Tm and GC content close to those of the forward primer is a good practice. 

Step 3 - As the template sequence runs in the 5' to 3' direction, you cannot order the reverse primer directly. The template sequence needs to be reverse complemented first. To do this, copy the sequence and paste it into a new window. Alternatively, select the primer sequence and click on the reverse complement button located in the header area of the APE tool. 

Step 4 – Verify that the sequence has been reverse complemented. Here’s how to do this - if the sequence is 5’-CTGGAGGACGGAAGAGGAAGTAA-3’ before reverse complementation, after reverse complementation, it should become 5’-TTACTTCCTCTTCCGTCCTCCAG-3’.

Please note, you must reverse complement the selected DNA sequence before providing it to a primer synthesizing company. If you fail to do this, you will end up with another forward primer that starts from the end of your gene of interest, which is of no use to you.