DAY 1: 6/6/17
I was super nervous for my first day and I didn't know what to really expect! Once I arrived, I was greeted by Dr. MacNeill, and we went through the plan for the week. He showed me around and where everything was in the lab. My first task was to read all the healthy and safety rules on my cubicle (yes, I got a whole cubicle desk for myself.. how cool is that?! super cool I say!). Once I read everything, I was ready to head to the lab, and feel like a true scientist.
Dr. MacNeill had prepared beforehand 4 plasmids (he attempted to make 5, but for some unknown reason, the 5th plasmid just wasn't having any of it, and decided not to work). Today in the lab, I attempted to mutate each binding site found in the cdc27 (p66 in humans) subunit. Plasmid 1 had its phosphorylation sites mutated. Plasmid 2 had one of its ubiquitylation sites mutated. Plasmid 3 had its sumoylation (SUMO) site mutated. Each plasmid was transformed into cdc27/cdc27del yeast diploid cells and incubated at 32oC for about a week (to allow cells to grow and colonies to form).
DAY 2: 7/6/17
Today's task was to cut, purify and ligate DNAs (plasmid sample and insert DNA), then transform E. coli. The plasmid used as N11 plasmid, and it was digested with NotI-HB restriction enzyme. The inner DNA cdc27 (spcdc27-HBT) alleles were also digested with NotI-HB restriction enzyme. Before ligating both the plasmid sample and the insert DNA together, their solutions were run in an agarose gel, against a DNA molecular weight tag, for comparison (see figure 1). Once ligation was done, a 50ul and a 150ul solutions were placed in LB+ ampicillin plates and insulated overnight, allowing transformation to occur.
NOTES: bacteria was used for transformation because it grows quicker than yeast.
From figure 1, it can be seen that the plasmid is A and that the insert DNA is B. This can be told, not only by being organised and labelling the solutions being placed in the agarose gel, but also, because of the molecular weight of both solutions. The insert DNA is smaller than the plasmid, which means it will travel further in the agarose gel.
DAY 3: 8/6/17
Today's task was to screen colonies by colony PCR and set up culture for mini-preps. However, one of the enzymes required for colony PCR hadn't arrived yet (it was still in Germany.. so we waited). However, I still did some lab work: the plasmids prepared on DAY 2 were grown, and 8 colonies were placed into 8x tubes containing LB Broth and ampicillin, and incubated in a shaker overnight to form bacterial culture.
NOTES: two primers will bind to the transformed plasmid, however, a signal will only be transmitted if both primers are pointed to each other (correct orientation). If it occurs that more than one insert DNA is present in the plasmid, then the shortest sequence will be amplified (correct orientation). The longer sequences can happen to be amplified, but they will mostly likely serve as a template for the amplification of the shortest sequence. Furthermore, the longer sequences are hard to be identified in an agarose gel.
DAY 4: 9/6/17
Today, the primers arrived, so it was possible to carry on colony PCR using the bacterial culture prepared on DAY 3. All the 8 PCR solutions were allowed to undergo 25 cycles in a PCR machine (took ~1 hour 15 mins), then solutions were run in an agarose gel against a DNA MW solution, for comparison. Solutions were placed in the gel in the order: DNA MW, 1, 2, 3, 4, 5, 6, 7, 8 (see figure 2 for results).
From figure 2, it can be seen that plasmid 3 and 7 have the strongest signal, suggesting that those two plasmids underwent transformation and ended up with the right sequence at the correct orientations. So, due to the results from figure 2, it was decided to prepare/transform plasmids 3 and 7 into cdc27/cdc27del diploid following the same protocol as in DAY 1.
DAY 20: 24/07/17 The results from DAY 19's PCR is shown in figure 16. It can be seen from figu...
DAY 1: 6/6/17 I was super nervous for my first day and I didn't know what to really expect! Once I arrived, I was greeted by Dr. MacNe...
This summer, I have secured an internship for the Summer of 2017 in the School of Biology at the University of St Andrews, under the supe...
DAY 5: 12/06/17 Today's plan was to confirm whether plasmids 3 and 7, in fact, contained the insert at the correct orientation. This wi...