As I stated in Last weeks entry, I intend to introduce specific mutation into the Cx32 protein. This will be done by using specially designed primers and a template plasmid containing the Wild-type Cx32 protein to produce several fragments which will be arranged and joined during the gibson assembly.
Making the primers
To make the primers the wild type Cx32 sequence was obtained and the residues i wish to mutate were identified. once identified the nucleotide sequence of these residues I intend to mutate were changed. A region of around 40 base-pairs around the mutations were selected and adjusted to ensure the binding temperature of all the primers are similar to ensure successful PCRs. An example of the process can be seen below:
WT sequence: AAA AAT GCG GTA AAA CTA AGC
Lys Asn Ala Val Lys Leu Ser
Mutation in primer: AAA AAT GGG GTA AAA CTA AGC
Lys Asn Gly Val Lys Leu Ser
These primers were used alongside a template plasmid containing the wild-type Cx32 to produce two fragments containing the first mutation. As the postition of the mutation was known i could calculate the length of the fragments. I, therefore, ran the PCR product on a Gel electrophoresis, seen below, to ensure the PCR was successful.
As can be seen two fragments of around 400 and 500 bp can be seen, which was expected and allowed me to proceed. Before proceeding, I also measured and recorded the concentrations of the fragments using the nanodrop.
With this procedure completed, I could move onto the next procedure, The Gibson Assembly. Before this I needed to remove the wild-type Cx32 from the plasmid, awaiting the introduction of mutated construct. This was simply done via a restiction digest. My template plasmid had a Kpn1 restiction site at either side of the Cx32 coding region so this process was straight-forward. Then, using the the concnetration measured earlier I calculated the necesary volume of each fragment and the restricted plasmid in order to perform the gibson assembly the compete the first mutation. The length of the construct was again measured using Gel electrophoresis, showing the construct was slightly over 900bp as expected, allowing the inference that the process was successful.
Overall, the first week in Laboratory was successful. After a long absence from the lab, due to the Covid-19 Pandemic, it was exciting to get reaquatinted with basic skills such as pippetting. Furthermore I have learnt some new and widely applicable techniques such as PCR and Gibson Assembly.
Comentarios