Primer Design for qPCR

Finding Known Primer Sets

• Check papers, especially supplementary tables
• Check PrimerBank http://pga.mgh.harvard.edu/primerbank/index.html

Making Your Own Primers From Entrez

1. Check if the primer is available in the lab using the Primer Data sheet at https://docs.google.com/spreadsheets/d/1kQ1G52XuoqpAWHIWT5H2UDz4aDhP285oUyYcj7XTB24/edit?ts=5925e7a2#gid=0. If the primers are not available, proceed to the next steps
2. Find your RNA sequence on entrez by starting with the gene at http://www.ncbi.nlm.nih.gov/gene (make sure its the correct species). Then pick the mRNA you want to probe, based on the isoform structure on that page. Click on that nucleotide. (it will be an NM number)
3. Under analyse this sequence click Pick Primers
4. Under PCR Product Size pick 70-150 as the range
5. Under Exon/intron selection -> Intron Inclusion check the box by Primer pair must be separated by at least one intron on the corresponding genomic DNA
6. If you want to look at a specific region of the mRNA and an appropriate primer pair in that region is not selected, change the range under PCR template on the top
7. Click Get Primers on the bottom and wait for results
8. Print this out, for future reference and when you order primers name them as follows species-Gene-direction-seq.start-seq.end, for example mm-SREBF1-FWD-127-254.
9. Enter both primers in the Primer Data sheet used in Step 1