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or too low Primer annealing temperature is typically 50-60C (may be higher or lower based on primer sequence and buffer components). Suggestion: Determine Tm/annealing temperature based on one of the following equations: If primers are 20-35 bases If primers are 14 - 70 bases Tp = 22 + 1.46(Ln)
Ln = 2(# G or C) + (# A or T)
TP = Effective annealing
temperature 2 - 5 Tm = 81.5 + 16.6 (log10 [J+]) + 0.41
(% G + C) - (600/l) - 0.063
(% Formamide) + 3 to 12
[J+] = concentration of Monovalent cations
l = length of oligo

Back to Top Primers are degraded or not optimal Primers should have same number A & T's versus G & C's, and they should be at least 14 bases for specificity. Suggestions:

  • If primers are short and A-T rich, add 0.9 - 2.0%(v/v) DMSO
  • If primers are G-C rich, add 1-10% (v/v) Formamide
  • Double check priming sequence, use primer design program if available
  • Check aliquot of primers on a gel to ensure they are not degraded
Back to Top Incomplete template denaturat
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