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From 2010.igem.org

Site Directed Mutagenesis

Reaction

• 5ul of 10x reaction buffer
  
• Xul (10ng) of dsDNA template
  
• Xul (125ng) of oligonucleotide #1
  
• Xul (125ng) of oligonucleotide #2
  
• 1ul of dNTP mix
• 3ul of Quiksolution
• ddH20 to a final volume of 50ul

PCR

TABLE

DPN1 Digest

• Add 1ul to the PCR producy and incubate at 37 degrees for 1 hour.

Transformation

• Gently thaw the competent cells on ice. Aliquot 100μL of the competent cells into a pre-chilled 14ml BD falcon polypropylene round-bottom tube.
• Transfer 2μL of the Dpn I-treated DNA from each control and sample reaction to separate aliquots of the competent cells. Swirl the transformation reactions gently to mix and incubate the reactions at 2ºC for 30 minutes.
• Heat pulse the transformation reactions for 45 seconds at 42º C and then place the reactions on ice for 2 minutes
• Add 0.5mL of NZY+ broth preheated to 42ºC and incubate the transformation reactions at 37ºC for 1 hour with shaking at 225-250 rpm
• Plate 50μL and 100μL volumes of the transformation reaction on plates containing the appropriate antibiotic for the plasmid vector.