IGEM:University of Chicago/2009/Notebook/Organophosphate Hydrolase/2009/10/05

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Removal of Duplicate Restriction Sites Mutagenesis 50 μL reactions: 5 μL accubuffer 5 μL 10mM dNTPs 5 μL Forward Primer 5 μL Reverse Primer 1 μL template 28 μL ddH2O 1 μL accusure Cycle: 95°C for 10 minutes repeat 12x: 95°C for 30 seconds 58°C for 30 seconds 68°C for 10 minutes ice for 2 minutes digest with 1μL dpnI for 1 hr Transform into d5 α cells Tube labels: 1. pRS314 + primers 314x reverse/forward 2. pRS316 + primers 316x reverse/forward 3. pRS314 + primers 300 reverse/forward 4. pRS316 + primers 300 reverse/forward 314x removes SpeI 316x removes PstI 300 removes XbaI/SpeI Accuprime pfx Reaction Done as a backup to previous reaction 50 μL reactions: 5 μL buffer 5 μL dNTPs 5 μL forward primer 5 μL reverse primer 28 μL ddH2O 1 μL accuprime Cycle 94°C for 2 minutes repeat 13x: 94°C for 30 seconds 55°C for 30 seconds 68°C for 5 minutes Leave overnight KOD reaction Done as a tertiary backup, but not likely going to be used 50 μL reactions: 5 μL buffer 1 μL dNTPs 5 μL forward primer 5 μL reverse primer 1 or 5 μL template 28 μL ddH2O 1 μL KOD Cycle: 94°C for 2 minutes Repeat 5x: 98°C for 10 seconds 68°C for 5 minutes hold at 12°C indefinitely