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PS1 PCR with PrP Control (12/8/2009) This worked well using the MJ Research PCR Machine and Andy’s homemade Taq Polymerase. Reagent Volume/rxn (ul) ddH2O 37.0 10X PCR Buffer 5.0 50mM MgCl2 2.5 5mM dNTPs 1 50M 237F 0.2 50M PrPa 0.2 50M Prps 0.1 Taq Polymerase 1 Tail DNA 3 PCR Program, MJ Research PTC-200 (called PS1 in MJ Research Machine) 1. 94C for 3 minutes 2. A. 94C for 30 seconds B. 64.0C for 1 minute C. 72C for 1 minute D. 40 Cycles A-C 3. 72C for 4 minutes 4. Hold at 4C Primer Sequences 237F: CAGGTGGTGGAGCAAGATG PrP-Antisense-J: AGCCTAGACCACGAGAATGC PrP-Sense-J: GACTATGTGGACTGATGTCGG Gel Percentage: 2% Expected Products: PS1 Transgenic animals should produce one band from the PS1 product at approximately 1.2 kb. Expected Products: Endogenous PrP All animals, transgenic and wild-type, should exhibit the 750 bp band, indicating successful amplification of the tail DNA.
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