Examples given are suited to TaqMan primer design for mouse genes.
- Search Entrez for the nucleotide sequence of interest. In the Limits sections, select Only From: RefSeq.
- Choose the result from the appropriate organism (Mus musculus). Copy the RefSeq Accession Number (probably begins with NM_).
- Enter the RefSeq Accession Number into ELXR, and select the appropriate organism (Mouse). This aligns the cDNA against the genomic DNA sequence, and produces a graphical representation of the distribution of exons.
- ELXR also generates primers for amplifying specific exons, using the genomic sequence of the introns on either side of the desired exon. To amplify across exons takes a bit more work:
- At the bottom of the ELXR output, select "View mRNA/EST to genomic sequence alignment ". This shows the exon boundaries in the cDNA sequence.
- Copy the cDNA sequence from Entrez, and paste it into Primer3. Select an appropriate mispriming library (Rodent + Simple). In the Targets field, add numbers that represent one of the exon boundaries. For example, if EXLR showed exons at 1-154 and 155-237, you'd enter '153,4' as a target.
- Set an optimal product size (200bp), primer size (20bp), primer Tm (60ËšC), primer GC content (50%), and adjust the minimum and maximum values to be close to these optimum values.
- Press 'Pick Primers', and the best primer pair should be shown with a graphical representation of their placement on the cDNA sequence. A number of additional oligonucleotides are also suggested.
- If ordering custom primers from Invitrogen, sequences can be entered and ordered online.