A week in The Kuo Lab

I don’t have a typical day but I do have a typical week in Kuo Lab. I conduct a thorough mutagenesis experiment where I complete multiple different steps and ultimately mutate the desired sites of the gene.

I start with preparing agar plates (plates that have the necessary conditions for bacteria to breed) and store them in the cold room (4 °C) for later use. Then I prepare my PCR samples by aliquoting the necessary reagents and template DNA plasmid. I add primers – specifically designed to mutate the desired codons of the amino acids – and run the PCR samples in a thermocycler where they get amplified. After that, I run a gel electrophoresis to check whether the reaction actually worked. Having confirmed that PCRs worked just fine, I use kinase, ligase and Dpn1 enzymes to convert the linear DNA product to circular (because bacteria would chop up the newly transformed DNA if it were linear) and get rid of the original non-mutated  templates. Now our products are ready for transformation. I start transforming the mutated DNA plasmid into bacteria to further increase the number of the plasmids. I thaw the frozen bacteria and aliquot the PCR products into the bacteria tubes. I incubate the bacteria in ice for 30 minutes and then do a heat shock by placing the tubes into the heat block at 42 °C for 30 seconds. This helps transformation since the pores in bacteria enlarge and take up the plasmid DNA more easily. After this step, I place the bacteria to agar plates and do an overnight culture to grow bacteria colonies.

Next day, I look at my plates first thing in the morning. If some of the plates don’t have any colonies, I start over for them and redo the previous steps (I know, kind of frustrating). For the ones that worked, I pick three colonies from each plate and prepare them for plasmid DNA purification by doing an overnight liquid culture. The next day I do a miniprep to purify the mutated plasmid DNA. I add buffers and centrifuge the cells multiple times in order to digest them and precipitate the unwanted biomolecules (fats, carbohydrates, proteins, RNA and bacterial DNA). After that I use a spectrophotometer to measure the concentration of the DNA. To make sure that my plasmid DNAs have the mutated gene insert, I use restriction enzymes and run another gel electrophoresis. After confirming, I send my samples for sequencing and get the results the next day.

Of course, none of this process goes smoothly. When I started my project the first week, I thought I would be done with this part of the project (generating mutated plasmid DNA) in two weeks. I can now see how naive I was. During these steps, we had to readjust the temperatures for thermocyclers multiple times to get a better yield from PCR and ensure transformation. We had to add new chemicals to PCRs because some of our primers had rich G-C nucleotide contents which made them bind to the other strand very strongly and disrupted the PCR. Sometimes everything seemed to work and we sent the plasmids for sequencing, only to find out that the original template DNA was transformed not the mutated ones. So, we had to restart everything and wait for Dpn1 enzyme to operate much longer to thoroughly get rid of the original template DNA. I will start my fifth week tomorrow and I still need two more mutations to proceed to the final part of my project. Although there were times I got really frustrated about my experiments, these experiences have taught me that troubleshooting is very important in scientific process and we have to be very resilient to reach success. I am pretty sure that my Western Blots won’t go as planned either and I’ll have to perform a lot of them. But I believe I will be able to overcome the hardships I encounter by trying new adjustments and not giving up until I succeed.

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