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:
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.