( Mg 2 + from 1.55.0 mM (in 0.5 ...)HotStarTaq PCR Handbook

Mg²+ from 1.55.0 mM (in 0.5 mM steps) using the 25 mM MgCl2 solution provided (see Table 2). 6. Primer concentration not optimal or
primers degraded Repeat the PCR with different primer concen trations from 0.10.5 M of each primer (in 0.1 M steps). In particular, when performing highly sensitive PCR check for possible degradation of the primers on a denaturing polyacrylamide gel. 7. Primer design not optimal Review primer design

Appendix

1. Starting template

Both the quality and quantity of nucleic acid starting template affect PCR, in particular the sensitivity and efficiency of amplification.*

Quality of starting template

Since PCR consists of multiple rounds of enzymatic reactions, it is more sensitive to impurities such as proteins, phenol/chloroform, salts, ethanol, EDTA, and other chemical solvents than single-step enzyme-catalyzed processes. QIAGEN offers a complete range of nucleic acid preparation systems, ensuring the highest-quality templates for PCR, for example the QIAprep system for rapid plasmid purification, the QIAamp and DNeasy systems for rapid purification of genomic
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