Designing primers

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
Line 6: Line 6:
*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 15:36, 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

Personal tools