Designing primers

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(Useful Primer Design Tools)
(Useful Primer Design Tools)
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==Useful Primer Design Tools==
==Useful Primer Design Tools==
*[http://synbio.mit.edu/tools/clipboard.cgi Austin's clipboard tool] - online tool for generating the complement, reverse complement and restriction enzyme site analysis of a DNA sequence.  It also translates the sequence and gives the amino acids properties.
*[http://synbio.mit.edu/tools/clipboard.cgi Austin's clipboard tool] - online tool for generating the complement, reverse complement and restriction enzyme site analysis of a DNA sequence.  It also translates the sequence and gives the amino acids properties.
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*[http://scitools.idtdna.com/scitools/Applications/OligoAnalyzer/|IDT Oligo Analyzer]] - A relatively complete suite of online tools for primer analysis.
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*[http://scitools.idtdna.com/scitools/Applications/OligoAnalyzer/|IDT Oligo Analyzer] - A relatively complete suite of online tools for primer analysis.
*[https://catalog.invitrogen.com/index.cfm Invitrogen]
*[https://catalog.invitrogen.com/index.cfm Invitrogen]
*[http://www.bioinformatics.vg/biolinks/bioinformatics/verbose/PCR%2520and%2520Primer%2520Design.shtml List of Primer Design Software]
*[http://www.bioinformatics.vg/biolinks/bioinformatics/verbose/PCR%2520and%2520Primer%2520Design.shtml List of Primer Design Software]

Revision as of 18:14, 21 July 2005

This needs more work, but wanted to get it started.

General Rules

  • Avoid runs over 3 nucleotide (AAAA)
  • 18-30bp in length. Molecular Cloning says that 5' tails do not significantly affect annealing.
  • Primer pairs should differ in length by less than 3bp.
  • 3’ end should be G or C (stronger bond)
  • Primer melting temp (Tm) should be 50-60C w/ low FIR difference (<5C, 2C better)
  • Molecular Cloning advises GC content between 40% and 60%
  • Avoid palindromes and inverted repeat sequences.
  • Avoid complementarity between members of a primer pair.
  • Check for dimer binding and hairpins in Vector NTI.
    • Want to avoid structures with ΔG < -5kcal/mol

BioBrick Primers

To BioBrick a part, the following tails should be added to your primers:

  • PREFIX Primer        cctttctagag        11 bp
  • SUFFIX Primer        tactagtagcggccgctgcagcctt        25 bp


The prefix primer adds an Xba site, and the suffix adds the entire BB suffix (Spe-Not-Pst)
Check the annealing temperature both without the tail (the first cycle or so) and with the tail (the later cycles).

Useful Primer Design Tools

Personal tools