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