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Technical Report #3

A site-directed mutagenesis reaction was carried out using thermal cycling to mutate a glutamate residue to a glycine in a plasmid using two designed primers. The PCR mixture was prepared and underwent temperature cycling and DpnI digestion. The reaction was transformed into E. coli cells which were plated, cultured, and isolated colonies were selected for plasmid purification. The purified plasmid was sequenced and the mutation was verified.

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18 views

Technical Report #3

A site-directed mutagenesis reaction was carried out using thermal cycling to mutate a glutamate residue to a glycine in a plasmid using two designed primers. The PCR mixture was prepared and underwent temperature cycling and DpnI digestion. The reaction was transformed into E. coli cells which were plated, cultured, and isolated colonies were selected for plasmid purification. The purified plasmid was sequenced and the mutation was verified.

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Alex Coronado
Copyright
© © All Rights Reserved
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Download as DOCX, PDF, TXT or read online on Scribd
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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

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