Designing primers: Difference between revisions
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This needs more work, but wanted to get it started. | This needs more work, but wanted to get it started. | ||
==General Rules== | |||
*Avoid runs over 3 nucleotide (AAAA) | *Avoid runs over 3 nucleotide (AAAA) | ||
*18-30bp in length | *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) | *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) | ||
*30-80% GC (>50% is better) | *30-80% GC (>50% is better) ([[Molecular Cloning]] advises between 40% and 60%) | ||
*avoid palindromes | *avoid palindromes and inverted repeat sequences. | ||
*check for dimer binding and hairpins in Vector NTI | *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 | **want to avoid structures with ΔG < -5kcal/mol | ||
==Useful Primer Design Tools== | |||
*VectorNTI | |||
*[https://catalog.invitrogen.com/index.cfm Invitrogen] |
Revision as of 11:50, 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
- VectorNTI
- Invitrogen