Maldi tof

Extension Primers were synthesized at Inqaba Biotechnical Industries Pty Ltd. Pretoria South Africa. They were diluted to a stock concentration of 500 µM. This stock was split into a four-tier concentration grouping of 7µM, 9µM, 11µM and 14µM according to extension primer mass from smallest to largest. This four-tier system was used for Oligovalidation and peak optimisation on the MALDI-TOF Then, the iPLEX extend was carried out with a final concentration of between 0.625 and 1.5 l µM for each extension primer, depending on the mass of the probe, iPLEX termination mix (Agena Biosciences) and 1.35µM iPLEX enzyme (Agena Biosciences) and conducted a two-step cycles program; 94 o C for 30 s followed by 40 cycles of 94 o C for 5 s, then followed 5 cycles of 52 o C for 5 s, and 80 o C for 5 s within the 40 cycles, then 72 o C for 3 min in the 40 cycles. The reaction was then desalted by addition of 6 mg resin to each well followed by mixing and centrifugation to settle the contents of the tube. The extension product was spotted onto a 96-well spectrochip before being flown in the MALDI-TOF (Matrix -Assisted Laser Desorption Ionisation Time of Flight) mass spectrometer (Agena Biosciences).