Designing primers

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This needs more work, but wanted to get it started.
This needs more work, but wanted to get it started.
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Some general rules:
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==General Rules==
*Avoid runs over 3 nucleotide (AAAA)
*Avoid runs over 3 nucleotide (AAAA)
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*18-30bp in length (what about tails?)
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*18-30bp in length.  [[Molecular Cloning]] says that 5' tails do not significantly affect annealing.
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*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)  
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*30-80% GC (>50% is better)
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*30-80% GC (>50% is better) ([[Molecular Cloning]] advises between 40% and 60%)
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*avoid palindromes
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*avoid palindromes and inverted repeat sequences.
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*check for dimer binding and hairpins in Vector NTI
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*Avod complementarity between members of a primer pair.
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*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
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==Useful Primer Design Tools==
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*VectorNTI
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*[https://catalog.invitrogen.com/index.cfm Invitrogen]

Revision as of 13:50, 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)
  • 30-80% GC (>50% is better) (Molecular Cloning advises between 40% and 60%)
  • avoid palindromes and inverted repeat sequences.
  • Avod 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