Primer Design - Be Your Own Designer!

PCR primers

While there are a number of ways to obtain a set of PCR primers to amplify your GOI, sometimes, you might just need to customize your own to zero in on a particular region to specifically amplify a transcript variant. The following method is a timeless protocol for those who like to design their own primers.

Preparation

First, you will need the cDNA sequence of your GOI. The sequence should ideally have the intron-exon boundaries clearly marked. For this I recommend getting the cDNA sequence from Ensembl as they have an option that allows users to mark the exons using an alternating color scheme (see below).

Screen capture of the exon sequences from the ABCA1 transcript variant 1 from Ensembl

Alternatively, you could just get the cDNA sequence and mark out the intron-exon boundaries yourself.

Ideally, you will want your primers to cover an intron-exon boundary. Crossing an intron-exon boundary will ensure that your primers are annealing to and only amplifying from your cDNA template rather than gDNA contamination. If your primers can anneal to both cDNA and gDNA, on an agarose gel, the gDNA will either give you a higher MW band, become a smear, or be the exact same size as your cDNA PCR product. Either way, gDNA contamination will affect quantification and subsequent analyses.

Primer design:

* You will want your primers to be ~20 nucleotides long and to have ~50% GC.

* Do not have strings of >3 nucleotides of any kind (e.g. AAAAA or GGG or TTTT or CCCCC).

* At the 5’ end, start with A or T nucleotides; at the 3’ end, have G or C nucleotides as the last 3 nucleotides. This will give a stronger bond to the 3’ end; you do not want the 5’ end to have too tight a bond.

* Aim for a product size of ~200bp.

* When ordering, standard desalt purification is fine for PCR and cloning.