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 :)
Thursday 8 July 2010
Day 9
It looks like my first experiment is a success! the colonies pictured below from yesterday (looking like a smudge in the top right corner) just happened to be the colonies which seem to grow on hygromycin (the new resistance gene) and dont grown on G418 anymore which probably means they have lost thier old resistance gene and that it has been replaced with the new one!
The first thing I did today was to prepare the colonies for fluctuation tests to measure how different the altered chromosome is in comparison with the wildtype in regards to how stable they are. I spread colonies across more plates in oder to get lone colonies as these are needed for running fluctuation tests.
After this I carried on with the other fluctuation test i am running and it is still running uncontaminated. I diluted samples ready for plating tommorow.
I then finished my DNA plugs from yesterday. They are now ready for analysis via pulse field gel. This is to find isochromosomes in the samples by sorting by size.
The first thing I did today was to prepare the colonies for fluctuation tests to measure how different the altered chromosome is in comparison with the wildtype in regards to how stable they are. I spread colonies across more plates in oder to get lone colonies as these are needed for running fluctuation tests.
After this I carried on with the other fluctuation test i am running and it is still running uncontaminated. I diluted samples ready for plating tommorow.
I then finished my DNA plugs from yesterday. They are now ready for analysis via pulse field gel. This is to find isochromosomes in the samples by sorting by size.
Wednesday 7 July 2010
Day 8
Today I was pleased to see some of the 75 colonies I spread on new plates had grown! I made replica plates of these growths on YEA (normal medium), the hygromycin antibiotic (they should now be able to grow on this) and the G418 (they should not be able to grow on this anymore if the transformation was completely successful). This should be the last selection stage. The photo below shows a plate with a yeast growth in the top right:
After putting the new selection plates in the incubator I then had to think about the other experiments i'd been given to do from other scientists investigations (about isochromosomes). I had to carry on the fluctuation test I began yesterday and make some more DNA plugs.
I first made sure that the cultures I made for the fluctuation test were infact yeast and not some other contaminant. I'll need to do this after each generation of the culture to make sure I am not diluting and growing something I shouldnt be. It would be a big waste of time. I then diluted the colonies so that they will carry on growing (have space) and put them back into the incubator.
After that I carried out most of the procedure for making DNA plugs though I didnt have time to finish, so I left the DNA at a safe point to finish tommorow morning. It is still very nice feeling to be doing these new experiments on my own.
After putting the new selection plates in the incubator I then had to think about the other experiments i'd been given to do from other scientists investigations (about isochromosomes). I had to carry on the fluctuation test I began yesterday and make some more DNA plugs.
I first made sure that the cultures I made for the fluctuation test were infact yeast and not some other contaminant. I'll need to do this after each generation of the culture to make sure I am not diluting and growing something I shouldnt be. It would be a big waste of time. I then diluted the colonies so that they will carry on growing (have space) and put them back into the incubator.
After that I carried out most of the procedure for making DNA plugs though I didnt have time to finish, so I left the DNA at a safe point to finish tommorow morning. It is still very nice feeling to be doing these new experiments on my own.
Tuesday 6 July 2010
Day 7
Today was quite a busy day. I was able to help other people in the lab with their experiments aswell as my own.
There isnt a lot to report about my own experiment. I had a look at the plates I began to grow yesterday but there wasnt really any good signs of growth and so I put them back to grow another night, I really hope something grows!!!
Apart from that I got to help and carry out parts of the procedure for making "DNA plugs". DNA plugs are literally DNA encased in agarose gel. In this form the DNA can be stored and then used in further experiments. In the interest of our group further experiments will probably be to view the remaining structures of chromsomes to see which parts have been lost (which markers etc). This procedure was very interesting, it involved using a microscope to count cells to make sure there were enough in each culture sample, some centrifugation, cell lysis and finally creation of the agarose plugs, its a procedure ive never heard of before working here and its a really good idea.
Ive also begun running a fluctuation test, its on a different experiment to my own, like the DNA plugs were but it was good practice for when I hopefully get to do my own. Ive begun by making up the 14 cultures to be left over night, they are my 7 wild type and 7 mutant samples. I will be taking cell number measurements from both in correlation to their generation number and working out mutation rates.
Tommorow I will hopefully be carrying on with my main experiment and also the fluctuation tests.
There isnt a lot to report about my own experiment. I had a look at the plates I began to grow yesterday but there wasnt really any good signs of growth and so I put them back to grow another night, I really hope something grows!!!
Apart from that I got to help and carry out parts of the procedure for making "DNA plugs". DNA plugs are literally DNA encased in agarose gel. In this form the DNA can be stored and then used in further experiments. In the interest of our group further experiments will probably be to view the remaining structures of chromsomes to see which parts have been lost (which markers etc). This procedure was very interesting, it involved using a microscope to count cells to make sure there were enough in each culture sample, some centrifugation, cell lysis and finally creation of the agarose plugs, its a procedure ive never heard of before working here and its a really good idea.
Ive also begun running a fluctuation test, its on a different experiment to my own, like the DNA plugs were but it was good practice for when I hopefully get to do my own. Ive begun by making up the 14 cultures to be left over night, they are my 7 wild type and 7 mutant samples. I will be taking cell number measurements from both in correlation to their generation number and working out mutation rates.
Tommorow I will hopefully be carrying on with my main experiment and also the fluctuation tests.
Monday 5 July 2010
Day 6
Today I was pleased to see that over the weekend a lot more colonies had grown on my antibiotic plates.
The colonies I'd picked the previous week however hadnt grown very well. My supervisor Felcity said that the colonies that the colonies that develop later on the plate are usually more likley to be what we are looking for. Especially as one of the first colonies to spring up was a contaminent.
As there were lots of colonies I spent a while picking more colonies to be spread on new plates. I made three plates with 25 colonies on each. Hopefully i'll have more success with some of those in future. There will be more chance of finding the right colony with 75 of them than with just the 4.
I was given some pipettes of my own to use. I tested them to make sure they were accurate by weighing the contents of their tips and checking it was the correct weight for the amount taken in.
After that, I begun to look into the next steps of the experiment as there was such a large amount of colonies growing and so more chance of finding the yeast colonies I want this time. I will be doing fluctuation tests on the yeast with the changed marker in comparison with the wild type yeast. Fluctuation tests as I said the other day are a measure of the rate of spontaneous mutation in bacteria. I will be running these to see whether there is a difference between the wild type and the chromosome with the new marker. This should then give way to more tests involving the creation of DNA "plugs" (DNA of the cells enacsed in agarose) which we may be able to use to show the exact form of the transformed chromosome to see if everything is where it should be. If everything is where it should be then the yeast strain is ready to use in chromosome observation experiments.
The fluctuation tests involve some statistics and will take a reasonable amount of time to carry out so I plan to read more about them so I know exactly what im doing. Having to repeat fluctuation tests if it goes wrong could take weeks.
Tommorow I will be going in early to watch how to make DNA plugs!
The colonies I'd picked the previous week however hadnt grown very well. My supervisor Felcity said that the colonies that the colonies that develop later on the plate are usually more likley to be what we are looking for. Especially as one of the first colonies to spring up was a contaminent.
As there were lots of colonies I spent a while picking more colonies to be spread on new plates. I made three plates with 25 colonies on each. Hopefully i'll have more success with some of those in future. There will be more chance of finding the right colony with 75 of them than with just the 4.
I was given some pipettes of my own to use. I tested them to make sure they were accurate by weighing the contents of their tips and checking it was the correct weight for the amount taken in.
After that, I begun to look into the next steps of the experiment as there was such a large amount of colonies growing and so more chance of finding the yeast colonies I want this time. I will be doing fluctuation tests on the yeast with the changed marker in comparison with the wild type yeast. Fluctuation tests as I said the other day are a measure of the rate of spontaneous mutation in bacteria. I will be running these to see whether there is a difference between the wild type and the chromosome with the new marker. This should then give way to more tests involving the creation of DNA "plugs" (DNA of the cells enacsed in agarose) which we may be able to use to show the exact form of the transformed chromosome to see if everything is where it should be. If everything is where it should be then the yeast strain is ready to use in chromosome observation experiments.
The fluctuation tests involve some statistics and will take a reasonable amount of time to carry out so I plan to read more about them so I know exactly what im doing. Having to repeat fluctuation tests if it goes wrong could take weeks.
Tommorow I will be going in early to watch how to make DNA plugs!
Friday 2 July 2010
Day 5
This morning I was pleased to find some colonies growing on one of the plates with the antibiotic on! This meant either, my experiment had worked and there were transformed yeast growing or some other contaminent was growing on it.
I was then shown how to make agarose plates containing more of the antibiotic. I was to pick some of the colonies and transfer some cells from each onto new plates, I also needed some more to replica plate the back up tranformation I began yesterday.
I spread cells from the 4 most visible colonies onto a new plate, I then decided to look at them under a microscope to make sure of what they were before moving on. It turned out that 3 out ofthe 4 colonies were yeast! The 4th however contained yeast and another smaller, stranger type of cell. This means my experiment may have worked in 3/4 cases and that I may have transformed yeast. I will have to discard the fourth however.
Once more colonies have grown over the weekend I will take some pictures. For now, the below diagram shows again what should have happened in the transformation. The old marker (G418) should have been replaced by a new marker (Hph). This was possible due to the upstream and downstream sequences of both being the same and so one should be able to replace the other.
There is still however a possibility that even if the transformation worked in the way that the new marker is now installed, that there is a chance the old marker has not been replaced. The old marker needs to be replaced for the further experiments taking place with this mini chromosome. To make sure it has been replaced another selection plate will be made up to see if the yeast will still grow on G418. If they do this means the yeast have both markers and the transformation did not work as we'd hoped.
Apart from this discovery I also got to watch some fluctuation tests, I think I understand them a bit better now. I will be doing them in future to observe rates of chromosome mutation like isochromosome formation.
There will be more on the new colonies and what they've turned out to be on monday!
I was then shown how to make agarose plates containing more of the antibiotic. I was to pick some of the colonies and transfer some cells from each onto new plates, I also needed some more to replica plate the back up tranformation I began yesterday.
I spread cells from the 4 most visible colonies onto a new plate, I then decided to look at them under a microscope to make sure of what they were before moving on. It turned out that 3 out ofthe 4 colonies were yeast! The 4th however contained yeast and another smaller, stranger type of cell. This means my experiment may have worked in 3/4 cases and that I may have transformed yeast. I will have to discard the fourth however.
Once more colonies have grown over the weekend I will take some pictures. For now, the below diagram shows again what should have happened in the transformation. The old marker (G418) should have been replaced by a new marker (Hph). This was possible due to the upstream and downstream sequences of both being the same and so one should be able to replace the other.
There is still however a possibility that even if the transformation worked in the way that the new marker is now installed, that there is a chance the old marker has not been replaced. The old marker needs to be replaced for the further experiments taking place with this mini chromosome. To make sure it has been replaced another selection plate will be made up to see if the yeast will still grow on G418. If they do this means the yeast have both markers and the transformation did not work as we'd hoped.
Apart from this discovery I also got to watch some fluctuation tests, I think I understand them a bit better now. I will be doing them in future to observe rates of chromosome mutation like isochromosome formation.
There will be more on the new colonies and what they've turned out to be on monday!
Thursday 1 July 2010
Day 4
Still no conclusive evidence of my experiment working today. After looking at the cultures it does look like there is some activity (or at least I like to think there is :P) tommrow will be two days after the replica plating, I hope to see some colonies tommrow!!
I spent the rest of the day setting up several more transformations, just incase this first one didnt work. It was good practice, it was good to do experiments by myself. In between doing this I also spent more time working on writing up my methods and trying to make sense of all the steps. I also got to watch more of the western blott procedure I saw some of yesterday.
Other than that I also learned a bit more about the process scientists go through to get their papers published.
I spent the rest of the day setting up several more transformations, just incase this first one didnt work. It was good practice, it was good to do experiments by myself. In between doing this I also spent more time working on writing up my methods and trying to make sense of all the steps. I also got to watch more of the western blott procedure I saw some of yesterday.
Other than that I also learned a bit more about the process scientists go through to get their papers published.
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