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RP-PCR Traces

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posted on 2023-07-14, 16:59 authored by Clarissa RoccaClarissa Rocca, Mariam Kekenadze, Valentina TurchettiValentina Turchetti, Sara NagySara Nagy, Nana Kvirkvelia, Maia Beridze, Rauan KaiyrzhanovRauan Kaiyrzhanov, Henry HouldenHenry Houlden

C9orf72 expanions tested by RP-PCR. Traces were generated by running the PCR products through the ABI 3730 machine for capillary electrophoresis.  A PCR Mastermix was prepared by mixing 12.5ul of Amplitaq Gold 360 Master Mix (ThermoFisher), 9.5ul of 5M Betaine (ThermoFisher), 1 ul of 10pmol/ul FAM labelled Forward primer (5'-TGTAAAACGACGGCCAGTCAAGGAGGGAAACAACCGCAGCC-3'), 1 ul of 10pmol/ul Reverse primer (5'-CAGGAAACAGCTATGACC-3'),1ul of 10pmol/ul repeat specific reverse primer (5'-CAGGAAACAGCTATGACCGGGCCCGCCCCGACCACGCCCCGGCCCCGGCCCCGG-3') and 1ul of 100ng/ul DNA. Samples were amplified with an initial heat denaturation of 95°C for 10 minutes, followed by 10 cycles of 95°C for 30 seconds, 58°C for 2 minutes, 72°C for 2 minutes, and then 25 cycles of 95°C for 10 minutes, followed by 10 cycles of 95°C for 30 seconds, 58°C for 2 minutes, 72°C for 2 minutes with a 20seconds increase per cycle. The final extension step was 72°C for 7 minutes. The PCR was run on a 9700 Block, at ramp speed 9600. After PCR, 1ul of the reaction product was addedd to a mix with 9.2ul of Formamide (Roche) and 0.1 ul of GeneScan 500 LIZ Size Standard (ThermoFisher).  

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    UCL Queen Square Institute of Neurology

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