Difference between revisions of "Primer Design for qPCR"
From Bridges Lab Protocols
Davebridges (Talk | contribs) (added direction to primer name) |
Davebridges (Talk | contribs) (updated specifying to start with the ncbi gene page) |
||
| Line 4: | Line 4: | ||
==Making Your Own Primers From Entrez== | ==Making Your Own Primers From Entrez== | ||
| − | # Find your RNA sequence on entrez by | + | # 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 |
# Under analyse this sequence click '''Pick Primers''' | # Under analyse this sequence click '''Pick Primers''' | ||
# Under PCR Product Size pick 70-150 as the range | # Under PCR Product Size pick 70-150 as the range | ||
Revision as of 14:51, 31 July 2014
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
- 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
- Under analyse this sequence click Pick Primers
- Under PCR Product Size pick 70-150 as the range
- 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
- 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
- Click Get Primers on the bottom and wait for results
- 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.
- Enter both primers in ExperimentDB
