Technical Report #3

Site-directed Mutagenesis
The site-directed mutagenesis reaction was carried out with thermal cycling. Two
reverse-compliment oligonucleotide primers were designed by Finch et. al. (2009)
that mutate glutamate GAG to glycine GGG. The two primer sequences are 5GGCATCGTGGCTGGGGTCCTGGTGTTG-3 and 5CAACACCAGGACCCCAGCCACGATGCC-3.
The PCR mixture was prepared in 50 L total volume using 5 L of 10x reaction
buffer, 5 L of plasmid (pET102-BasTM), 1 L of forward primer, 1 L of reverse
primer, 1 L of dNTP mix, 3 L of QuikSolution, and 1 L of PfuUltra DNA
polymerase. For the first cycling segment, the reaction went through 1 cycle
incubating at 95*C for 1 minute. In the second segment, it went through 18 cycles
incubating for 50 seconds at 95*C, then 50 seconds at 60*C, and then 5 minutes at
68*C. The third segment included 1 cycle incubating at 68*C for 7 minutes. After
temperature cycling, restriction enzyme Dpn I (1 L) was added and the reaction
incubated at 37*C for 1 hour.
The site-directed mutagenesis reaction (3 L) was transformed into 50 L of XL10Gold E. coli cells by incubating on ice for 15 minutes, and then at 42*C for 30
seconds. Then, 250 L of SOC medium was added to the cells and they were
incubated at 37*C while shaking at 200 rpm for 1 hour. The cells were then spread
on an agar plate containing carbenecillin and incubated overnight at 37*C. Isolated
colonies were then picked off the plate and dropped into LB medium.
For plasmid purification, 1.5 mL of culture were centrifuged at 13,000 rpm for 1
minute. After centrifugation, the supernatant was discarded and the pellet was
resuspended in 250 L of P1 buffer. Then, 250 L of P2 buffer was added and the

solution was mixed by inversion. Finally, 350 L of N3 buffer was added and the
solution was mixed. After centrifuging for 10 minutes at 13,000 rpm, 750 L of
supernatant was transferred into a new tube. Into the new tube containing the
supernatant, 750 L of cold 100% ethanol was added and then centrifuged at
13,000 rpm for 10 minutes. After discarding the supernatant, 500 L of cold 70%
ethanol was added and then centrifuged at 13,000 rpm for 5 minutes. After
discarding the supernatant, pellet was resuspended in 50 L of MiliQ water. For
absorbance reading, a 1:20 dilution of the plasmid was made and the plate was
read at 260 nm. A sequence for the mutated plasmid was also determined and
verified.
q-RT-PCR