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Trouble-shooting: Low yield

href="#Option13">Evaporation Primer annealing temperature is too high

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 Template not clean or degraded

For example, protease contamination can degrade the polymerase.

Suggestions:

  1. Try "Hot start" technique
  2. Purify the DNA template to the highest degree possible, including proteinase K digest
Back to Top An<
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