Cultured cells were obtained (American Type Culture Collection, Manassas, VA). Qiagen Incorporated?s ?Generation Capture Column Kit? (Qiagen Incorporated, Valencia, CA) was used to extract DNA from cultured cells (Qiagen 2010). An Epoch Multi-Volume Spectrophotometer System (BioTek, Winooski, VT) was used to determine the purity of the DNA (BioTek 2010).

Primers were designed to detect the R702W mutation in the human genome.  The primers contained intentional mismatches to avoid indiscriminate binding (Yaku et al 2008).  Reaction cocktails were mixed in thermocycler tubes; each contained 40 of μL H20, 5 μL of 10X PCR buffer, 1 μL of 10mM dNTPs, 1 µL of 100µM appropriate forward and reverse primer, 1 µL of genomic DNA template, and 1 µL of Taq polymerase (Invitrogen, Carlsbad, CA). Each sample was centrifuged at 5000 rpm for 60 seconds. DNA amplification was performed in a GeneAmp thermal cycler (Applied Biosystems, Foster City, CA).  Per cycle, the denaturing conditions were 94°C for 30 seconds; the annealing conditions were 54°C for 45 seconds, and the extension conditions were 72°C for 45 seconds.  An initial denaturizing stage of 94°C for five minutes was used and a final extension condition of 72°C for five minutes.  A total of 30 cycles were performed.

A TBE agarose gel was made (Invitrogen 2010). The agarose gel was submerged in 1X TBE buffer solution in an electrophoresis case. 10mL of appropriate samples were pipetted into agarose gel wells. 5 µL of 1 Kb Invitrogen DNA Ladder was added to the first well; a control well consisting of processed, DNA-free master mix was added to the final well. Electricity was applied to the electrophoresis case for approximately 25 minutes at 136 volts.

Gels were analyzed by ultraviolet transmission using a Kodak Gel Logic system (Carestream Health, Woodbridge, CT). Bands were analyzed at the 1000 base pair position as indicated by the ladder. Bands at the 1000 base pair position in a wild type test indicated wild type DNA; bands at 1000 base pairs in a mutant type test indicated mutant type DNA. Both wild type and mutant type primers were not expected to anneal to the same DNA template in a single test. Any contradiction to this was considered a faulty test. Any test with bands appearing in the control lane was considered a contaminated set.