Table 1 1.3(Continued) To Enhance This Manipulate One or More of These Componen infirm that template is unnicked, free of contaminants and inhibi Use a smaller size template DNA (get more molecules per pg of input template, and less complexity for primer annealing). For example, PCR product vs genomic DNA Decrease amplicon size. Taq>Pfu, >>Stoffel fragment. Decrease cycling time or use a shuttle profile( Cha et al., 1992) Decrease the size of the tube Check for tight fit between reaction vessels and heating block. Decrease ramp time Use forward and reverse primers that have milar length and gC content. nfirm that primers do not form primer dimer or hairpin structure Reproducibility Ensure that template is clean and intact onfirm presence of sufficient starting template and sufficient sample numbe for statistical analysis. Use the same lots of primer and buffers between experiments. Store enzyme in small aliquots. for presence of contaminating template and inhibitors to PCR reaction. trols Include positive and negative controls with all experiment Cyclin Use a hot-start strategy( Kellogg et al Use the same cycler between experiments. Quantitative Confirm the quantity of the template Confirm template preparation is clean. Investigate for presence of contaminating template and inhibitors to PCR reaction Experimental design Include triplicate or quadruplicate samples Use a statistically sufficient number of sam repare a standard curve to demonstrate the range over which PCR product yield provides a reliable measure of the Robust: Confirm that chemistry, primer design, tubes, thermal cycler, and other factors are optimized. Confirm the analytical methods accuracy/resoluton. Is it accurate during the exponential phase of PCR? 298 Aoyagi298 Aoyagi Table 11.3 (Continued) To Enhance This Parameter Manipulate One or More of These Components Template Confirm that template is unnicked, free of contaminants and inhibitors. Use a smaller size template DNA (get more molecules per pg of input template, and less complexity for primer annealing). For example, PCR product vs. genomic DNA. Decrease amplicon size. Enzymes Taq > Pfu, >>Stoffel fragment. Cycling Decrease cycling time or use a shuttle profile (Cha et al., 1992). Decrease the size of the reaction tube. Check for tight fit between reaction vessels and heating block. Cycler Decrease ramp time. Primer design Use forward and reverse primers that have similar length and GC content. Confirm that primers do not form primer￾dimer or hairpin structure. Reproducibility Sample Ensure that template is clean and intact. Confirm presence of sufficient starting template and sufficient sample number for statistical analysis. Reagents Use the same lots of primer and buffers between experiments. Store enzyme in small aliquots. Investigate for presence of contaminating template and inhibitors to PCR reaction. Controls Include positive and negative controls with all experiments. Cycling Use a hot-start strategy (Kellogg et al., 1994). Use the same cycler between experiments. Quantitative Template Confirm the quantity of the template. Confirm template preparation is clean. Investigate for presence of contaminating template and inhibitors to PCR reaction. Experimental design Include triplicate or quadruplicate samples. Use a statistically sufficient number of samples. Prepare a standard curve to demonstrate the range over which PCR product yield provides a reliable measure of the template input. Robust: Confirm that chemistry, primer design, tubes, thermal cycler, and other factors are optimized. Analysis Confirm the analytical method’s accuracy/resoluton. Is it accurate during the exponential phase of PCR?