Today began with inspecting the replica plates I made of the mutant pli-1 strain. I had to mark colonies that were present on all replica plates because this means they should have all three of the markers we wish to be present (inculding the minichromosome with new hph gene). I took scrapings of these colonies to streak onto more plates for further selection.
After that I looked at my new hph/wt fluctuation test and diluted the samples. I made plates for plating my fluctuation test on tommorow, this time ive definetly made the right plates! I was a bit nervous about boiling the agar again, but im much more careful now.
I spent the rest of the day making DNA plugs, I made two sets of 16. I also began another set which i will finish tommorow.
Tuesday 20 July 2010
Monday 19 July 2010
Day 16
Firstly I had a look at the last stage of my fluctuation tests (the plates that had been growing over the weekend). On inspection the Pli-1/wild type fluctuation test seemed to have worked, there were a similar number of colonies growing on each plate. The Hph/wt test however hadnt worked so well, there seemed to be an acceptable number of colonies growing of the Hph samples but not of the wildtype. I realised i'd made a mistake when making up the plate mediums and this is what caused the problem. I had to start this test again as it is no good without controls from the wild type to compare to.
I then went on to look at the Hph/Pli-1 strain I had streaked in a plate for replica plating. The colonies seemed to have grown well, so I replica plated them onto plates containing Hph and Nat and also a plate that is ura -. If a colony grows on all three of the replica plates it is likely that it contains all three of the markers we want.
Some colonies from the Hph and Pli-1 fluctuation tests were inoculated for growth over the next few days in order to be later lysed to be made into DNA plugs.
This means over the rest of the week I will be carrying on a new fluctuation test, making DNA plugs and running pulse field gels. There is also a seminar that looks interesting going on at the centre on wednesday which I might attend; on "The role of rad50 in protecting the genome".
I then went on to look at the Hph/Pli-1 strain I had streaked in a plate for replica plating. The colonies seemed to have grown well, so I replica plated them onto plates containing Hph and Nat and also a plate that is ura -. If a colony grows on all three of the replica plates it is likely that it contains all three of the markers we want.
Some colonies from the Hph and Pli-1 fluctuation tests were inoculated for growth over the next few days in order to be later lysed to be made into DNA plugs.
This means over the rest of the week I will be carrying on a new fluctuation test, making DNA plugs and running pulse field gels. There is also a seminar that looks interesting going on at the centre on wednesday which I might attend; on "The role of rad50 in protecting the genome".
Sunday 18 July 2010
Day 15
There wasnt alot to do in the lab today so I helped out with a few 'house keeping' tasks, such as transferring DNA plug samples into smaller containers to make more room in the storage area for the large number of plugs about to be made.
After that there wasnt much left to do except plan the week ahead, which looks to be very busy due to the amount of experiments going on. Other than that I have been putting together my methods so far for the book im going to put together.
The picture below is just some of the results from running my DNA plugs in the pulse field gel:
This photo is just showing the plugs i made, further left of these plugs there was a ladder run for comparison so that we could determine what each of the bands were. We could see which samples were the same weight as the normal mini chromosome and which were isochromosomes.
After that there wasnt much left to do except plan the week ahead, which looks to be very busy due to the amount of experiments going on. Other than that I have been putting together my methods so far for the book im going to put together.
The picture below is just some of the results from running my DNA plugs in the pulse field gel:
This photo is just showing the plugs i made, further left of these plugs there was a ladder run for comparison so that we could determine what each of the bands were. We could see which samples were the same weight as the normal mini chromosome and which were isochromosomes.
Thursday 15 July 2010
Day 14
Felt much better today, went in early and caught up with all my work. I diluted and plated all of the samples from my fluctuation tests. thye will be left to grow now for three days, hopefully they will work this time so I can produce some statistical results. The fluctuation test for the Pli 1 mutant will be added to previous tests to build up a large number of results to give a better idea of isochromosome frequency in comparison to wild type. The fluctuation test for the mini chromosome now with Hph will show whether it behaves differently from wild type so we can see what to expect when we use it for further experiments.
There was also a lab meeting, in this one of my collegues gave a talk about the project we are all working on concerning sumoylation. It was good to go over why we are doing all the experiments and see the big picture again.
The yeast I grew which should include the new mini chromosome with Hph marker was crossed with the Pli 1 mutant we wish to observe in further experiments with this marker. The resulting tetrads from the cross have been spread on further plates to select for progeny which has the double mutant we are looking for with all the required markers.
Lastly, I have been asked to write up a method book for the lab to make it easier to teach methods to more people in future and just for reference, so i'll also be writing that in my spare time.
There was also a lab meeting, in this one of my collegues gave a talk about the project we are all working on concerning sumoylation. It was good to go over why we are doing all the experiments and see the big picture again.
The yeast I grew which should include the new mini chromosome with Hph marker was crossed with the Pli 1 mutant we wish to observe in further experiments with this marker. The resulting tetrads from the cross have been spread on further plates to select for progeny which has the double mutant we are looking for with all the required markers.
Lastly, I have been asked to write up a method book for the lab to make it easier to teach methods to more people in future and just for reference, so i'll also be writing that in my spare time.
Day 12
Today (this was meant to be written tuesday) I had a bit of an accident involving agarose gel and hurt my hand so ive not written anything til now.
Before I managed the silly accident I managed most of my work for the day. I diluted my samples for both of my fluctuation tests.
I also got to view the results of the pulse field gel I began yesterday, I will add pictures to a later post as I havent yet scanned the photos. The pulse field gel was successful, it showed some bands at the right size to be the normal mini chromosome, it also showed some samples had formed isochromosomes. The bands were very clear which showed the DNA plugs were nicely formed.
I did not work on wednesday due to the accident, one of my collegues carried on my dilutions in the fluctuation test for me, thats all I had to do that day.
Before I managed the silly accident I managed most of my work for the day. I diluted my samples for both of my fluctuation tests.
I also got to view the results of the pulse field gel I began yesterday, I will add pictures to a later post as I havent yet scanned the photos. The pulse field gel was successful, it showed some bands at the right size to be the normal mini chromosome, it also showed some samples had formed isochromosomes. The bands were very clear which showed the DNA plugs were nicely formed.
I did not work on wednesday due to the accident, one of my collegues carried on my dilutions in the fluctuation test for me, thats all I had to do that day.
Monday 12 July 2010
Day 11
Unfortunetly I found out that today my fluctuation test did not work properly.. this was because the mutant strain seems to have lost its Ura marker and grew far too much, so much so that I could not count colonies on the plates. So I started the experiment again by inoculating again with the same strains grown on plates which should gurante they have not yet lost ura.
I also began another fluctuation test but this time on the product from my own experiment with the new marker, in order to see how the altered mini chromosome fares in comparision to the wild type.
I got to see how to run a pulse field gel using the DNA plugs I made the other day. Pulse field gels are similar to normal gel electrophoresis except that the electric current is not passed straight from one end to the other, it is run in more of a zigzag. This makes it possible to run DNA of much larger size in a gel e.g. our minichromosome. Hopefully tommorow I should get a good picture of the results.
I also began another fluctuation test but this time on the product from my own experiment with the new marker, in order to see how the altered mini chromosome fares in comparision to the wild type.
I got to see how to run a pulse field gel using the DNA plugs I made the other day. Pulse field gels are similar to normal gel electrophoresis except that the electric current is not passed straight from one end to the other, it is run in more of a zigzag. This makes it possible to run DNA of much larger size in a gel e.g. our minichromosome. Hopefully tommorow I should get a good picture of the results.
Friday 9 July 2010
Day 10
Today involved a lot of repetitive tasks, the second part of the fluctuation test involved firstly counting cells from each of the samples and then working out individual dilutions to make sure enough cells from each sample were plated. After this set of dilutions I made triplicates of each plate. I then needed to make further dilutions in order to plate just 500 cells onto the normal medium (luckily only 1 copy of these were needed for each sample). These plates will be left over the weekend for further counting on monday!
I then got to cross the yeast strain I made from my transformation (with the altered mini chromosome) into a mutant strain called p1-delete. This is to add the mini chromosome (with new hph resistance gene) into yeast which we can use with the Bioneer Library (a mutant library which uses S.Pombe yeast). This crossing should give us the mutant yeast with our new mini chromosome.
Lastly I learned about another metho of testing chromsome stability with the use of UV or gamma radiation and survival curves. I also will need to do many many more crossings (1000s) with this library and so I may be taught how to use the robot next week in order to do this quickly :)
I then got to cross the yeast strain I made from my transformation (with the altered mini chromosome) into a mutant strain called p1-delete. This is to add the mini chromosome (with new hph resistance gene) into yeast which we can use with the Bioneer Library (a mutant library which uses S.Pombe yeast). This crossing should give us the mutant yeast with our new mini chromosome.
Lastly I learned about another metho of testing chromsome stability with the use of UV or gamma radiation and survival curves. I also will need to do many many more crossings (1000s) with this library and so I may be taught how to use the robot next week in order to do this quickly :)
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