Thursday, 27 July 2017


DAY 10: 19/06/17
The western blot prepared on DAY 9 was ran and scanned. Image is shown below:


The results didn't show a clear protein purification and that could be, for instance, while loading the gel, solutions ran from one well to another, mixing things up. However, it was unclear why the protein wasn't fully purified. Independently of the results not being clear, it was a good practice to make a western blot myself as I had never done it before (at uni, when we used one we were given a 'bought' ready one).

Today, spores solutions for b3.4 and 3 transformed plasmids were prepared and incubated overnight at 32oC

DAY 11: 20/06/17
Today, the spores solutions prepared yesterday, DAY 10, were plated out on EMM + Histidine + Adenine plates, and all 16 solutions (4 colonies from each plasmid) from plasmids 1, 5, 8 and 10 were plated on YE4S plates (YE4S medium had ampicillin and chloramphenicol added to it, in order to avoid bacterial contamination).

INFO: YE4S medium is a rich medium, and therefore all fungi should grown in it. EMM, however, as mentioned before, is a minimal medium and only the cells adapted to overcome the nutrients deprivation in the medium will survive.

So, for now any further step in my experiments require waiting for results, waiting for cells to grow. Thus, tomorrow will be a day off. Whoop, whoop I will sleep much much later than I'm used to these past weeks! 😎

DAY 12: 22/06/17
Today all plates were analysed to see colony growth. Also, because I've been using so many plates, I've prepared some more YE4S plates, and also YE4S + 5FOA plates for future use.

Again, in order to proceed with my experiments, waiting time to allow cells to grow more was required, so the weekend will be pro-longed with tomorrow! 😁


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 will be done by preparing solutions of plasmid 3 and 7, and N1, analysing them and sending them to be sequenced in a lab in Dundee (that's nice right, to have a lab that will do all the sequencing for you, and it's not even that far away..all you need to do is to send/receive a post).

Solutions of N1, 3 and 7 plasmids were prepared by mixing the DNAs with enzyme EcoRV, water and 10x cut smart buffer. A loading dye was added to each solution to help the solutions to 'fall' into the agarose gel wells. The solutions were loaded in an agarose gel and ran, the results are shown in figure 3.


Results from figure 3 just seems odd, and even the DNA MW marker that we saw last week looks odd compared to figure 1 and 2. So, since there was 'extra' of the N1, 3 and 7 plasmids solutions, another gel was ran. The results are shown in figure 4.


This set of results looks much better, however, it can be noticed that solution 3 wasn't ran properly as it seems that the gel was 'damaged', possibly by the pipette while loading solution 3. For that reason, from now on, only solutions 7 and N1 will be analysed. Note, however, that we can't just tell whether the primers are in the right direction only by looking at this figure. So, all solutions were sent to be sequence (in Dundee.. remember I mentioned?). Once they've been sequenced, their sequence can be compared to the 'expected sequence' (N1).

While waiting for the sequencing to be back, spores solutions (haploid cells) were made from the plates prepared on DAY 1. About 10 colonies from each plate was resuspended in water and helicase enzyme, then incubated overnight at 32C. 

INFO: why helicase enzyme you may ask? well, helicase digest diploid yeast cells, and also digests the cell walls of ascus cells (cells with 4 spores present), releasing haploid cells (spores). Why are the ascus cells you may ask? Well, that's because the spores were grown on EMM. EMM plates are poor plates, where only yeast cells capable of producing all amino acids are able to grow. Also, at starving conditions, diploid yeast cels will undergo meiosis, forming ascus cells. Sometimes, the cell wall naturally breaks, releasing haploid cells.

MORE INFO: In day 1, 4 plasmids were transformed, as shown below:
1. leu1-32/leu1-32
2. Ura4-D18/Ura4-D18
3. his3-377/his3-377
4. ade6-M210/ade6-M216  
⇨ The yeast strand used in day 1 is sp280 cdc27+/cdc27::ura4+ (cdc27+/''cdc27Δ), where :: means replaced with.
⇨ It can be seen that leucine, uracil, histidine and adenine are unable to be made due to their gene's alleles mutation/deletion. So, how are these yeasts going to grow? Well..

  •   Plasmid 1 is fixed with Leu2 plasmid (leu2 ≣ leu1+), where ≣ means equivalent to. 
  •  Plasmid 2 is fixed with ura4+ in cdc27 
  •  Plasmid 3 is fixed by the addition of histidine in EMM 
  •  Plasmid 4 doesn't need to be fixed when diploid. This is because Ade6 is a dimeric protein, and as you can notice, different alleles of the same chromosome were deleted. Thus, ade+ is produced. However, in haploid cells, adenine is added to EMM to enable the yeast to obtain adenine and grow. 

DAY 6: 13/06/17
Today the spores solutions prepared yesterday, on DAY 5, will be placed on EMM + Histidine + Adenine plates. Histidine? the cell is unable to produce it, and adenine? spores are haploid, and therefore, an adenine- mutation on plasmid pHBLA-Cdc27-N8, deletion of a gene will stop adenine production (in diploid cells, due to being diploid, the dimeric protein having two different alleles of the same chromosome being deleted would still allow the production of adenine+). So in order for the cells to be able to grown in EMM plates in haploid form, both histidine and adenine are added to EMM. Each spore solution was plated as it is, but also diluted in 1/10, 1/100 dilutions.

So this all so far have been part of my project, but because my part of internship now requires a lot of waiting (e.g. wait for cells to grow). So I helped Dr. MacNeill with one of his projects by preparing plasmid solutions for 3 plasmids,  Z1, Z2 and Z8. Then prepared restrictive enzyme solutions, with EcoRV and HinIII enzymes, to run an agarose gel. This was good laboratory practice, as I had learn how to do this, so now I'm having the chance to emphasise my learning and put it in practice.

Two gels were ran because for the first one, a mistake occurred (yes I know... tragic!!). So, I mistook the loading dye (which helps solution to sink in the gel wells) with MyTaqRed (an element for PCR reactions). In my defence, they both had the same colours 😝. So I added MyTaqRed instead of loading dye, and when I realised my mistake (I realised although the liquid looks the same colour, pink/purple, the tubes are different... the MyTaqRed is not labeled and the loading dye is labelled), I added loading dye as well.. I knew that some ingredients that make up MyTaqRed wouldn't have affected the reaction, but I was afraid the TaqPolymerase enzyme would, so I repeated everything again. The Results are shown in figure 5 and 6. Figure 5 represents the first reaction with the addition of MyTaqRed and figure 6 represents the repeated version, without the MyTaqRed.



Figure 5 shows a better gel than figure 6. This is because, after all, the MyTaqRed, did not affect the results, but also it looks like the mixtures were better mixed, and the pipetting better carried out. This is concluded due to, in figure 6, the Z1 HindIII restrictive enzyme solution shows an extra band that shouldn't be there, suggesting that the enzyme didn't cut the plasmid properly. Also, the Z2 EcoRV restrictive enzyme solution shows that the gel was slightly damaged. Apart from that both gels look clear. Furthermore, to analyse the results, the bands sizes were compared to the DNA plasmids sequence, confirming that the plasmids solutions were correct. Sequences were analysed using Enzyme X program software.

DAY 7: 14/06/17
Today the sequences from the DNA plasmids 3 and 7 were analysed, to confirm whether they had the right insert at the right orientation. Also, plasmid b3.4 was transformed into diploid strain: pHBLA-Cdc27-Ne (with K187R mutation)

The sequences for DNA plasmids 3 and 7 were analysed using first '4 peaks' software, to analyse the quality of the peaks, and check where about the sequence start/stop to be clear. The ALIGN website was used to analyse the DNA plasmids sequence 3 and 7. Both sequences were compared to the expected sequences for a plasmid with an insertion in the right orientation, reverse orientation and a plasmid without the HBT insertion. After analysing the sequences, it was concluded that both plasmids (3 and 7) had the HBT insert at the right orientation (both identity and similarity shows 100% for the correct orientation.

Plasmid b3.4 was transformed into diploid strain: pHBLA-Cdc27-Ne (with K187R mutation), and transformed cells were plates and incubated at 32oC. This transformed plasmid is the plasmid that I didn't have on DAY 1 (2nd Uni binding site).

DAY 8: 15/06/17
Today we analysed under a light microscope how the spore plates prepared in DAY 6 and also the transformed plasmic pHBLA-Cdc27-N3 prepared in DAY 7, as well as plasmid 3.

INFO: Although the plates from DAY 6 still need to be left to grow for longer, it can be seen that the diluted plates show less and more spread out spores (haploid cells). It can also be seen that some plates show bacterial contamination, but we will worry about that later on, when the yeast colonies have had time to grow more.

Today a SDS gel was also prepared to be used tomorrow.

DAY 9: 16/06/17
Today I started the preparation of the SDS gel running/western blot. The protocol is long and even tho there is only two line written so far, it took me the whole day to complete it. The blot itself was allowed to dry to be used on Monday again.


DAY 20: 24/07/17 The results from DAY 19's PCR is shown in figure 16.                                       It can be seen from figu...