Difference between revisions of "Primer Design for qPCR"
From Bridges Lab Protocols
Davebridges (Talk | contribs) (updated specifying to start with the ncbi gene page) |
m (added a first step of checking the primer availability in the lab using the primer data sheet in google drive before ordering the primer and edited the last step as to where to add the new primers (on the primer data sheet)) |
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==Making Your Own Primers From Entrez== | ==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 | + | # 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 |
| + | #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 | ||
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# Click '''Get Primers''' on the bottom and wait for results | # 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'''. | # 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 | + | # Enter both primers in the Primer Data sheet used in Step 1 |
[[ Category: qPCR ]] | [[ Category: qPCR ]] | ||
Revision as of 20:39, 24 May 2017
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
- 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
- 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 the Primer Data sheet used in Step 1
