User:Omar Choudary/Notebook/Omar Choudary CHEM-571 AU-2011/2012/2011/09/20

Chem-571

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Objective Perform Polymerase Chain Reaction (PCR) to mutate Green Fluorescent Protein (GFP) to include a cysteine residue after the enterokinase cleavage site Method's for experiment were based off "QuickChange Site-Directed Mutagenesis Kit": [1] Description Primers were determined using the following link: [2] Reaction mixture was prepared using the following: 42 μL dH2O 5 μL 10x reaction buffer 1 μL dsDNA template (50 ng/μL) 1.25 μL Primer 1 1.25 μL Primer 2 1 μL dNTP mix (10 mM) 1 μL of PfuTurbo DNA Polymerase (2.5 Units/μL) was then added to reaction mixture 25 μL of Bio Rad liquid wax was added to the top of the mixture Reaction mixture was placed into thermocycler with the following cycles: 1 cycle 95 Degrees Celsius for 30 seconds 15 cycles as follows: - 30 sec at 95 Degrees Celsius - 1 min at 55 Degrees Celsius -3.6 min at 68 Degrees Celsius Final cycle 4 Degrees Celsius for 24 hours Data Theoretical Primers Forward Primer 5' - CGA CGA TGA CGA TAA GCG ATG GGG ATC CGA ATT CGC - 3' Reverse Primer 5' - GCG AAT TCG GAT CCC CAT CGC TTA TCG TCA TCG TCG - 3' 56% G/C Content Melting Temperature - Tm = 79.1 Degrees Celsius (Salt Adjusted) Actual Primers Forward Primer 5' - GAC GAT GAC GAT AAG GAT CGA TGG GGA TCC GAA - 3' Reverse Primer 5' - TTC GGA TCC CCA TCG ATC CTT ATC GTC ATC GTC - 3' Melting Temperature - Tm > 78 Degrees Celsius G/C % > 40% Notes The GFP plasmid was 3600 base pairs in length The mutation involved transforming the GAT codon located after the enterokinase clevage in the GFP plasmid. The vector used in this experiment is the PRSET/DNGFP vector. The mutation is a D1CGFP mutation.