Allele Calling Software 27. What can you suggest for setting up the instrument(s) and or measurement of FP?
• Proper filters must be installed in the instrument to measure the fluorescence of R110 (Ex 490 nm/Em 520 nm max) and TAMRA (Ex 550 nm/Em 570 nm max). Some instruments also require that the appropriate dichroic mirrors be installed. • For the Victor2 V the recommended filters are: • R110 485ex 535/25em • TAMRA 544ex 595/60em • Setting or adjusting of the “G” factor is highly recommended to facilitate troubleshooting and comparisons at different sites with different instruments. The G factor is a calibration that sets the mP reading of a solution of free terminator to 50. With the G-factor set to 1, read 20 L of solution containing 1 L of Terminator Mix and 2 L of 10X Reaction Buffer. Insert the S and P intensities into the following equation and calculate the G-factor for each dye. G = 950S/1050P • The FP value is calculated by following formula: mP = 1000 x [Ivv – Ivh]/[Ivv + Ivh] where Ivv is the emission intensity measured when the excitation and emission polarizers are parallel an
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- Allele Calling Software 27. What can you suggest for setting up the instrument(s) and or measurement of FP?
