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Different concentrations of a Taq polymerase were tested using primer mixture C (Fig. 32). The most efficient enzyme concentration seemed to be around 0.4µl or 2 Units/25µl reaction volume. Too much enzyme, possibly because of the high glycerol concentration in the stock solution, resulted in an unbalanced amplification of various loci and a slight increase in the background. too little enzyme resulted in the lack of some of the amplification products.
Fig. 32.Amplification products of mixture C, using 0.5 Units/25µl, 1 Unit/25µl, 2Units/25µl, 4 Units/25µl and 8 Units/25µl reaction volume are shown. Arrows indicate the expected positions of the amplification products. The most appropriate enzyme concentration was between 1-2 Units/25µl.
Five native Taq polymerases, from five different sources, were used to amplify multiplex mixture D in 1.6x PCR buffer using 2Units enzyme/25µl reaction (Fig. 33). In the same buffering conditions, all these enzyme performed similarly.
Fig. 33.Multiplex PCR of mixture D in 1.6x PCR buffer using Taq polymerases from five sources. Lanes 1 to 5 indicate that all enzymes work similarly at the same concentration. Lane 4* (green) shows the products obtained when the enzyme from lane 4 was used in the buffer provided by the vendor. An unspecific product appeared, indicating that buffer composition influences the results.