( ...)HotStarTaq PCR Handbook

1. Too much starting template Check the concentration and storage conditions of the starting template (see appendix). Make serial dilutions of template nucleic acid from stock solutions. Perform PCR using these serial dilutions. When re-amplifying a PCR product, start the re-amplification round using 1 l of a 1-in- 103104 dilution of the previous PCR. In most cases, a nested PCR approach results in higher specificity and sensitivity for reamplification (see appendix). 2. Carry-over contamination If the negative-control PCR (without template DNA) shows a PCR product or a smear, exchange all reagents. Use disposable pipet tips containing hydrophobic filters to minimize cross-contamination. Set up all reaction mixtures in an area separate from that used for DNA preparation or PCR product analysis. 3. Enzyme concentration too high When using HotStarTaq DNA Polymerase, use 2.5 units per 100 l reaction. When using HotStarTaq Master Mix, use 25 l Master Mix per 50 l reaction. 4. Too many cycles Reduce the number of cycles in steps of 3 cycles. 5. Mg²+ concentration not optimal Perform PCR with different final concentrations of
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