Designing primers: Difference between revisions

Revision as of 11:50, 23 May 2005

This needs more work, but wanted to get it started.

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

@@ Line 1: / Line 1: @@
 This needs more work, but wanted to get it started.
-Some general rules:
+==General Rules==
 *Avoid runs over 3 nucleotide (AAAA)
-*18-30bp in length (what about tails?)
+*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)
+*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 &Delta;G < -5kcal/mol
+==Useful Primer Design Tools==
+*VectorNTI
+*[https://catalog.invitrogen.com/index.cfm Invitrogen]