Designing primers: Difference between revisions
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*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% | ||
*avoid palindromes and inverted repeat sequences. | *avoid palindromes and inverted repeat sequences. | ||
*Avod complementarity between members of a primer pair. | *Avod complementarity between members of a primer pair. | ||
| Line 16: | Line 16: | ||
*[[VectorNTI]] | *[[VectorNTI]] | ||
*[https://catalog.invitrogen.com/index.cfm Invitrogen] | *[https://catalog.invitrogen.com/index.cfm Invitrogen] | ||
*[http://frodo.wi.mit.edu/primer3/primer3_code.html Primer3] | |||
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.
- 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
