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The amount of template in the reaction is not optimal The necessary amount of template varies from reaction to reaction. As a guideline, 100 - 750 ng human DNA (105 - 106 copies) per 100 l reaction. The amount of enzyme should be optimized for each template. Suggestions:
  1. Titrate the amount of template in the reaction
  2. Perform optimization experiment varying enzyme concentration by 0.50 increments in suggested range (0.5 to 5.0 units)
Back to Top An enzyme inhibitor is present in the reaction Suggestion: Reduce or remove the concentration of any inhibitor in the reaction mixture. Known inhibitors of PCR include: 50 mM ammonium chloride
EDTA (metal chelator)
> 0.8 M Hematin
PBS (phosphate will bind free magnesium)
> 0.02 % sarcosyl
0.5 M urea
> 5 % DMF
> 10 % formamide
heparin
> 20 % PEG deoxycholate
> 0.01 % SDS (can be reversed with equal molar ratio of NP40 and Tween 20)
> 10 % DMSO
> 20 % glycerol
> 0.4% N-Octylglucoside
Residual Phenol
>0.06% Sodium deoxycholate Back to Top Primer annealing temperature is too high
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