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PCR reaction 3 (nested PCR)

LP Livius Penter
MB Mehdi Borji
AN Adi Nagler
HL Haoxiang Lyu
WL Wesley S. Lu
NC Nicoletta Cieri
KM Katie Maurer
GO Giacomo Oliveira
AA Aziz M. Al’Khafaji
KG Kiran V. Garimella
SL Shuqiang Li
DN Donna S. Neuberg
JR Jerome Ritz
RS Robert J. Soiffer
JG Jacqueline S. Garcia
KL Kenneth J. Livak
CW Catherine J. Wu
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1 µl resuspended bead-bound PCR 2 product was amplified under qPCR control until end of exponential amplification with stop at end of elongation in a total volume of 50 µl. 25 µl Q5 High-Fidelity 2X Master Mix (New England Biolabs, Cat. no. M0492S), 5 µl 1:1,000 nuclease-free H2O pre-diluted 10,000x SYBR Green I Nucleic Acid Gel Stain (Invitrogen, Cat. no. S7563), 1.25 µl 20 µM CGAvenus.PS primer, 1.25 µl 20 µM each patient-specific nested primer mix. 98 °C 5 min (initialization), 98 °C 20 s (denaturation), 62 °C 30 sec (annealing), 72 °C 4 min (elongation). After purification of PCR products with 65 µl ProNex Beads (1.3x) (Promega, Cat. no. NG2002) and elution in 50 µl. Quantification of PCR products using TapeStation (Agilent, Cat. no. 5067–5365 and 5067–5366).

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