For PCR Primers:

  • Highlight the gene that you want to amplify
  • Select "Primers" from the top banner and "design new primers" from the drop down tab
  • Make sure you have checked "Forward Primer" and "Reverse Primer"
  • Set task to "precise" if you are doing this for a cloning reaction
  • check "Target Region"
  • Modify characteristics as needed - see checking Tm and other primer design tips in the protocols page
  • Hit OK and pick the best primer generated - hovering your mouse over the primer will give you Tm and GC content

For Sequencing Primers:

  • If your gene is less than 1500 base pairs you can sequence it using M13 Primers and do not need to design your own
  • If your gene is larger than 1500 base pairs:
  • Highlight a 50-100 bp region that you want the primer to bind in
  • Select "Primers" from the top banner and "design new primers" from the drop down tab
  • Set task to "Generic"
  • Check forward primer and DO NOT check reverse primer
  • Check "Included Region"
  • Set "Tm Min" to 50 optimal to 55 and "Tm Max" to 60
  • Set "GC Min" to 45 optimal to 50 and "GC Max to 55
  • Set "GC Clamp" to 1
  • Hit OK and pick the best primer generated - hovering your mouse over the primer will give you Tm and GC content

General tips for sequencing:

Reads are only clear about 30 bp after the primer to 600 bp after the primer.

The easiest way to pick primers is to start with one in a 50 bp region in front of the gene and then measure out 500 bp from that primer then place the next one in the following 100 bp region then measure out 500 bp and place one in the next 100 bp region and so on.

If you are planning on doing additional cloning with this part try to make some of the primers usable for future constructs

For Both:


  • Once your primers are designed. right click on the primer annotation and select "Extract Regions" this will make a primer file in the same folder
  • Select all of your primers and then click File => Export
  • Export the documents as a .csv
  • Highlight only name and sequence
  • Email the csv to your supervisor to be ordered

 

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