The results from DAY 19's PCR is shown in figure 16.
It can be seen from figure 16 that the PCR with primers NRP1-K185-REV didn't work on DNAs #5-1, #5-2, #6-3, #6-4, even ad adapted conditions (lower annealing temperature, 48oC). So, a new PCR was prepared with NRP1-HBT-REV primer mix instead. Results are shown in figure 17.
It can be seen from figure 17 that there is a very light DNA band being emitted for #6-3 and #6-4. However, it is not very clear, so a new set of results were ran with the resting PCR. This time the exposure was changed in order to try and see the DNA bonds more clearly. Results are shown in figure 18.
It can be seen from figure 18 that there are DNA bands, however, the signal is weak (hence the need to increase exposure). Figure 19 is the same results as shown in figure 18, but with lowered exposure, showing no DNA bands at all to the naked eye (I thought this was a cool thing to show.. how looking at things from different 'angles' can change your interpretation of things).
From figure 15, it can be seen that N5-3 and N5-4 give a nice DNA signal, so those 2 PCR were sent to be sequenced against HBS2 primer.
DAY 21: 25/07/17
Today I will practice to do serial dilutions and spotting assays using a 96 well plate and a pinning tool (apparently this tool is very expensive, so I shall have to be extra careful with it)
Dr. MacNeill prepared beforehand 3 yeast cultures: wild type, N5-1 and N5-3. In order to do the dilutions, the OD600nm of each culture was measured, giving wild type OD = 0.26, N5-1 = 0.24, and N5-3 OD = 0.23. As it can be seen, the OD values for all 3 yeast cultures are of a close range, so there was no need to dilute the cells to OD600nm of 0.1 (also, this is just a practice day). Once the OD values were figured out, the cell dilutions was prepared, and cells were pinned to YES4 plates using the pinning tool (exciting bit). Dr MacNeill showed me step by step how do all of this, because I will have to do it on my own from no on.
In preparation for tomorrow, I prepared a culture for N5-1 and wild type (WT) pre-cultures (previously prepared by Dr. MacNeill), and left it to grow overnight at a shaking bath at 30oC.
DAY 22: 26/07/17
Today I did everything I learnt yesterday (on my own) using the N5-1 and WT cultures I prepared yesterday. The plates were incubated at 20oC, 32oC and 35oC to see how temperature influences growth.
The OD600nm values for the WT and N5-1 cultures were 0.17 and 0.19, respectively. Therefore, there wasn't a need to dilute the solutions to an OD value of 0.1.
Today pre-cultures for WT, N1, N3, N5, N8 and N10 DNAs were prepared and allowed to grow overnight at 32oC.
DAY 23: 27/07/17
Today I made cultures from the pre-cultures prepared yesterday, and the cultures were allowed to born overnight at a 30oC bath with shaking.
The plates prepared on DAY 21 and 22 were analysed. They still need to be left to grow for longer, however, it was possible to see the effects of the dilutions and how well presented a plate is by using a pinning tool (instead of manually pipetting all solutions into the agar plate). See this photo so you know what I mean:
DAY 24: 28/07/17
So today I used the WT, N1, N3, N5, N8, and N10 yeast cultures, prepared yesterday, to do serial dilutions and spotting assays.
The OD600nm values for the WT, N3 and N8 yeast cultures were below 0.1, so they were allowed to grow for ~30/40 minutes longer in the shaking water bath at 30oC. Apart from culture N10, all the other cultures, N1, N5 had their OD values of 0.11 and 0.13 (slightly above from 0.1, the desired OD value). All cultures were adjusted to have an OD value of 0.1, either by diluting or letting the cells grow more. Once all cultures had their OD value of 0.1, the serial dilutions were prepared
The serial dilution solutions were pinned on YE4S plates + HU (hydroxyurea) plates, with different HU concentrations: 0, 3, 6, 9 and 12 mM. Solutions were also pinned on YE4S plates to undergo UV light treatment. The UV light wavelengths were 50, 100, 150, 200 and 250nm. All plates were incubated at 32oC.
INFO: The purpose of growing cell at 'damaging' conditions (hydroxyure, UV light) is to see if the mutated cells (N1, N3, N5, N8, N10) are able to grow and if so, when they stop being able to grow.
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