Published: 2011 ISBN: 978-1-904455-72-1
Price: GB £159 or US $310
An essential book for all scientists using PCR, real-time PCR, qPCR and related techniques. read more ...
Full details of this book at PCR Troubleshooting and Optimization. More figures at PCR Figures.
Chapter 1. Figure 8. Rapid-cycle, real-time PCR and melting analysis. Momentary denaturation and annealing allows amplification in 10-20 min. Fluorescence is acquired once each cycle and is used for detection and quantification. Melting analysis is performed immediately after PCR and is used for genotyping, product identification and/or heterozygote scanning. The real-time data was obtained on a Roche LightCycler 1.5 by amplifying a 250 bp fragment of exon 2 of PIGA from human genomic DNA in the presence of 1X LCGreen Plus dye. After a 5 s denaturation at 95 C, 45 cycles of 95 C for 0 s, 60 C for 0 s, and 72 C for 2 s with a 2 C/s ramp between annealing and extension temperatures was performed. Fluorescence was acquired at the end of each extension step. Temperature cycling required just over 15 min, while melting analysis at 0.2 C/s required another 4 min.
Further reading at PCR Troubleshooting and Optimization. More figures at PCR Figures.