Haynes Lab:Notebook/Investigating Photo-Switchable Synthetic Nucleosomes/2013/04/25

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April 25, 2013

  • QuickChange Site Directed Mutagenesis
  • Use 50 ng of dsDNA template and 125 ng of each primer. Template strands are about 5kb for H2B-GFP and 7kb for LOV.
Reagents H2B LOV
Plasmid DNA (50 ng) 0.4 μL 0.2
primer 1 (10 μM, 125 ng) 1.7 1.7
primer 2 (10 μM, 125 ng) 1.7 1.7
10x reaction buffer 5.0 5.0
dNTP mix 1.0 1.0
dH2O 40.2 40.4
Total 50.0 50.0
  • Add 1 μl of PfuTurbo DNA polymerase (2.5 U/μl) to each control and sample reaction
  • Thermal cycling
    • 95°C/30 sec
    • [95°C/30 sec; 55°C/1 min; 68°C/7 min (1 min/kb plasmid length)]x18
    • 4°C, ∞