As stated in the last blog post, the Construct containing the first mutation is completed and is ready to be transfected into cells. This was my first experience working with any kind of cell culture or transformations. The construct was transformed into competent HeLa cells. These cells were then plated onto ampicilin containing media to prevent any contamination, considering my construct contained an ampicilin resistance gene. This ensured that any bacteria that grow on the plate are those containing my construct. These were allowed to grow overnight. 15 colonies were then selected and isolated. The inclusion of my construct within each of these colonies was then confirmed via Gel electrophoresis.
From the Gel electrophoresis, it can be seen that colonies 1-13 all contain my construct, as they contain the expected 900bp fragment. The best colonies were selected for liquid culture and subsequent DNA extraction and isolation, providing me a pure isolate of my constructs DNA. To make sure that this mutation was correctly incorperation, the DNA sample was sent for sequencing.
Overall, whilst all the procedures carried out this week seemed to be successful I am awaiting the results of the sequencing. despite the procedures being completed, I still ran into some difficulties. Some PCRs did not run as expected, however it was a useful experience in trouble-shooting. After adjusting the annealing temperature and the extension time, I was able to successfully complete the PCR. I learnt that despite possible further optimisation, 63 degrees for annealing temperature and a 1 minute extension time work the majority of the time when using Q5 polymerase.
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