Designing primers: Difference between revisions
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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) | ||
*[[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 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== | ==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
