Difference between revisions of "Designing primers"

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*Primer pairs should differ in length by less than 3bp.
 
*Primer pairs should differ in length by less than 3bp.
 
*3’ end should be G or C (stronger bond)
 
*3’ end should be G or C (stronger bond)
*primer melting temp (Tm) should be 50-60C w/ low FIR difference (<5C, 2C better)  
+
*Primer melting temp (Tm) should be 50-60C w/ low FIR difference (<5C, 2C better)  
 
*[[Molecular Cloning]] advises GC content between 40% and 60%
 
*[[Molecular Cloning]] advises GC content between 40% and 60%
*avoid palindromes and inverted repeat sequences.
+
*Avoid palindromes and inverted repeat sequences.
*Avod complementarity between members of a primer pair.
+
*Avoid complementarity between members of a primer pair.
*check for dimer binding and hairpins in Vector NTI.
+
*Check for dimer binding and hairpins in Vector NTI.
**want to avoid structures with &Delta;G < -5kcal/mol
+
**Want to avoid structures with &Delta;G < -5kcal/mol
  
 
==Useful Primer Design Tools==
 
==Useful Primer Design Tools==

Revision as of 12:36, 23 May 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

Useful Primer Design Tools