To perform a PCR reaction to mutate green fluorescent protein (GFP) to contain a cysteine amino acid just after the enterokinase cleavage site.
Procedure derived from QuikChange® Site-Directed Mutagenesis Kit
- Synthesize and purify two complimentary primers containing the desired mutation.
- Prepare reaction mixture with 5 μl of 10× reaction buffer, 1 μL of dsDNA template (50 ng/μL), 1.25 μL of oligonucleotide control primer #1 (100 ng/μL), 1.25 μl of oligonucleotide control primer #2 (100 ng/μL), 1 μl of dNTP mix (10mM), and 41.75 μL of double-distilled water (ddH2O) to a final volume of 50 μL.
- Add 1 μL of PfuTurbo DNA polymerase (2.5 U/μL) to the reaction mixture.
- Add 25 μL Bio Rad Chill Out™ liquid wax.
- Place reaction in thermocycler, for 1 cycle at 95°C for 30 s.
- Keep reaction mixture in thermocycler for 16 cycles as follows:
- 30 s at 95°C
- 1 min at 55°C
- 3.6 min at 68°C
- Leave reaction mixture in thermocycler at 4°C for 24 hr.
Primer determined using http://www.basic.northwestern.edu/biotools/oligocalc.html:
- 5'- GAT AAG GAT GAC GAT AAG TGT CGA TGG GGA TCC GAA TTC GCC -3'
- 5'- GGC GAA TTC GGA TCC CCA TCG ACA CTT ATC GTC ATC CTT ATC -3'
- 39bp, 51% GC content, Tm=78.8°C (salt adjusted).
Primer actually used in experiment:
- 5'- GAC GAT GAC GAT AAG GAT CGA TGG GGA TCC GAA -3'
- 5'- TTC GGA TCC CCA TCG ATC CTT ATC GTC ATC GTC -3'
Primers must have a melting point Tm ≥ 78°C, a GC content ≥ 40%, and be between 25-45 bases in length.
Sequence of GFP vector provided by Invitrogen.