Welcome back everyone. It seems like I had only just started researching down at the lab, but it has finally come to an end. This week was the final week at my internship and I am sad to see it come to an end. Thankfully, I was successful in my endeavors to synthesis a thermostable DNA polymerase, in this case pfu, but there was still so much more I could have done with it. Creating the pfu was only the first step, there was still so much more testing I could have done, whether it be comparing it to an actual company made pfu or figuring out more specifics for my pfu. Nonetheless, I still succeeded and had fun.
Nothing terribly exciting happened this week. I worked more on my presentation, constantly changing little things hear and there, but for the most part, it is done (glad to have that off my shoulders). Other than me continually presenting, nothing else really happened. There was one event of interest though. I finally gave someone else in the lab a sample of my pfu for them to test in their own experiment. Unfortunately, I was not able to hear how well it performed before I left; I hope it worked well for them.
But, it is with a heavy heart that I say this blog and my project are nearing their ends. Thank you all for reading up until this point, if you have just found this blog, I hope you find it interesting! And with that, I bid you all farewell.
Saturday, April 25, 2015
Saturday, April 18, 2015
The Final Tests
Welcome back everyone! This week is my second to last one at my internship and was primarily dedicated to getting ready to present about my project, but before I could do that, I had to do one last PCR.
This PCR was also finally able to settle that adding BSA to the PCR solution does not change the amount of DNA produced.
It is unfortunate that it does not increase efficiency, but the simpler it is to run the better.
The rest of my time was spent working on my presentation and creating a PowerPoint detailing my project to help me better explain what I did. Hopefully, I have done a good job with that in this blog so far, so it should not be that hard to finish my presentation up. In other news, I found out exactly how much money I saved the lab by creating this pfu. But before I was able to calculate that, I had to find out just how much pfu I made (I know that I made 24mL, but how concentrated is that 24mL). By comparing my pfu's effectiveness side-by-side with taq, I was able to determine that .25μL of pfu was about .125μL of taq. 0.125μL of taq is .625 units according to the manufacturer. Now you may be thinking what exactly a unit is, and that is where a slight problem comes up. Each manufacturer classifies units a different way, but for the sake of convenience, I will just use this taq for my unit comparison. Since I used .625 units of taq, and my pfu produced pretty much the same amount, then it is safe to say that I used was about .625 units of pfu. This would mean that my pfu has 2.5 units/μL. And this is where it gets a little sketchy, I need to compare my pfu with a store bought equivalent, which my lab unfortunately does not have. So, I ended it up just comparing my pfu to another pfu product I found online, which means the money that I saved may not be perfectly accurate. Enough beating around the bush, I found that a total of 96,000 reactions could be performed with my pfu. When compared to a store bought pfu, it was $133 for 160 reactions. This means that I made $79,800 worth of pfu by only spending like $300. That is a lot of money! Too bad I can't sell it and make a fortune; still needs to be regulated and requires much more thorough inspection. I think it is safe to say that this project was a success! Next week will be my final post, so stay tuned!
 |
| This was to demonstrate the difference in the amount of DNA produced to the amount of pfu used |
 |
| There is barely any difference between these two. |
The rest of my time was spent working on my presentation and creating a PowerPoint detailing my project to help me better explain what I did. Hopefully, I have done a good job with that in this blog so far, so it should not be that hard to finish my presentation up. In other news, I found out exactly how much money I saved the lab by creating this pfu. But before I was able to calculate that, I had to find out just how much pfu I made (I know that I made 24mL, but how concentrated is that 24mL). By comparing my pfu's effectiveness side-by-side with taq, I was able to determine that .25μL of pfu was about .125μL of taq. 0.125μL of taq is .625 units according to the manufacturer. Now you may be thinking what exactly a unit is, and that is where a slight problem comes up. Each manufacturer classifies units a different way, but for the sake of convenience, I will just use this taq for my unit comparison. Since I used .625 units of taq, and my pfu produced pretty much the same amount, then it is safe to say that I used was about .625 units of pfu. This would mean that my pfu has 2.5 units/μL. And this is where it gets a little sketchy, I need to compare my pfu with a store bought equivalent, which my lab unfortunately does not have. So, I ended it up just comparing my pfu to another pfu product I found online, which means the money that I saved may not be perfectly accurate. Enough beating around the bush, I found that a total of 96,000 reactions could be performed with my pfu. When compared to a store bought pfu, it was $133 for 160 reactions. This means that I made $79,800 worth of pfu by only spending like $300. That is a lot of money! Too bad I can't sell it and make a fortune; still needs to be regulated and requires much more thorough inspection. I think it is safe to say that this project was a success! Next week will be my final post, so stay tuned!
Saturday, April 11, 2015
Additional Testing
Welcome back everyone! This week was all about testing, re-testing, and even more testing of the pfu I created. Of course, this testing involved the use of PCR, which can be quite aggravating to set up, but some more definitive results are starting to appear. I have managed to find the proper concentration of my pfu for PCR, so all that remains is finding other possible buffers to make it run more efficiently or adding other proteins to make the pfu more stable.
As for the proper concentration, I have found that .25µL of my stock pfu works very well in 25µL of solution. I have even been able to compare these results with that of taq polymerase (another thermostable DNA polymerase and the one my project was originally going to be about) and they seem about even in the amount of DNA produced through PCR, see my last post for a picture.
I have now been experimenting with different buffers, which did not work at all, but we kind of scrapped the idea of using new buffers as my first one seems to work just fine. So, now we are experimenting with adding a protein to the solution called BSA, Bovine Serum Albumin. What BSA is meant to do is provide stability to the pfu which causes it to function better. Unfortunately, for all the PCR tests I have done with it, I have gotten pretty inconsistent results, whether that's my fault (which it probably is) or some other problem, its hard to say. Sometimes it works far better than without it, other times it makes the PCR not work at all. I will likely test this again next week, to find out for certain if it really does do anything.
Since I do not have any very interesting pictures other than a ton of pictures of all the different PCRs I have done, I thought I might show you what the bench I work at looks like.
And in case you were curious, this is what it looks like to run a gel electrophoresis as that is how I know how well my PCR turned out.
I am nearing the end of this project and I thank all of you for reading it up until now, if you haven't well better late than never! See you all next week!
As for the proper concentration, I have found that .25µL of my stock pfu works very well in 25µL of solution. I have even been able to compare these results with that of taq polymerase (another thermostable DNA polymerase and the one my project was originally going to be about) and they seem about even in the amount of DNA produced through PCR, see my last post for a picture.
I have now been experimenting with different buffers, which did not work at all, but we kind of scrapped the idea of using new buffers as my first one seems to work just fine. So, now we are experimenting with adding a protein to the solution called BSA, Bovine Serum Albumin. What BSA is meant to do is provide stability to the pfu which causes it to function better. Unfortunately, for all the PCR tests I have done with it, I have gotten pretty inconsistent results, whether that's my fault (which it probably is) or some other problem, its hard to say. Sometimes it works far better than without it, other times it makes the PCR not work at all. I will likely test this again next week, to find out for certain if it really does do anything.
Since I do not have any very interesting pictures other than a ton of pictures of all the different PCRs I have done, I thought I might show you what the bench I work at looks like.
 |
| Ooooooooh Science! |
 |
| More Science! |
Saturday, April 4, 2015
The Results are In!
Welcome back everyone. This week was a monumental one. A culmination
of all the work had I done in the prior weeks. I was finally able to
test my pfu polymerase in a PCR reaction. I find it a little funny that I
have not done PCR once this whole project up until now considering my
entire project was based around it, but what would I have done with it
before now?
Some of you may be wondering, what is PCR? Simply put, it is polymerase chain reaction, which makes a lot of sense if you think about it. The whole goal of PCR is to replicate mass quantities of a template strand of DNA or a specific section of the template through the use of primers, DNA polymerase, and nucleic acids. A general summary of PCR is that the DNA is heated so that it unwinds and splits apart, the primers anneal to their complementary sites on the DNA (so like adenine to guanine and thymine to cytosine), the DNA polymerase then attaches to the primer and starts to move down the strand attaching complementary nucleic acids until it hits a stop, and then it repeats that around 30 more times, effectively doubling the amount of DNA every time. Of course, we can't see any of this happening so how do we know it worked? With a gel of course!
Before I could start the PCR though, I had to check the purity of my pfu solution.
Now,
I was finally ready to start PCR, and it was so, so very frustrating.
There were just too many tiny tubes and even smaller amount of liquid
that keeping check of them in an ice bucket was a nightmare. But, I got
all of tubes ready, slapped them in the thermocycler, and let that baby
run for 3 hours.
The next day, I came in, and ran a gel.
I
never really expected my pfu to work and neither did my mentor. What I
find to be even more strange is that other researchers are now asking if
they can use it, but I still need to run some more tests before that.
To think that I am nearing the end of my project with good results is
incredible, hopefully nothing catastrophic happens, though I think I am
in the clear for now. Stay tuned in next week for some more definitive
analysis of my pfu!
Some of you may be wondering, what is PCR? Simply put, it is polymerase chain reaction, which makes a lot of sense if you think about it. The whole goal of PCR is to replicate mass quantities of a template strand of DNA or a specific section of the template through the use of primers, DNA polymerase, and nucleic acids. A general summary of PCR is that the DNA is heated so that it unwinds and splits apart, the primers anneal to their complementary sites on the DNA (so like adenine to guanine and thymine to cytosine), the DNA polymerase then attaches to the primer and starts to move down the strand attaching complementary nucleic acids until it hits a stop, and then it repeats that around 30 more times, effectively doubling the amount of DNA every time. Of course, we can't see any of this happening so how do we know it worked? With a gel of course!
Before I could start the PCR though, I had to check the purity of my pfu solution.
 |
It looks more pure than last time, the
other stuff in it will not affect anything, probably.
|
The next day, I came in, and ran a gel.
 |
I FLIPPING DID IT! Pfu is in order of
decreasing concentration (strange because the last column has more
DNA than the one before it). Taq was used as a control to test how
well my pfu worked.
|
Saturday, March 21, 2015
Final Purification?
Welcome back everyone! As you know from last week, I created a 1L batch of my pfu producing Bl21, sonicated it, and centrifuged it, but it still contained DNA leftover from the lysed E. Coli. If we were to use the solution I created for PCR in this state, it would work in replicating whatever DNA we want (if we put in the right primers and such, more on that next week), but it would also replicate the contaminating E. Coli DNA and separating DNA can be a pretty difficult and expensive process. Now, how do we get rid of this contaminating DNA? Well, its actually very simple. All we had to do was treat the pfu solution with an enzyme called DNase I. DNase I, when put in the proper buffer, will indiscriminately cleave DNA leaving it in very tiny fragments. These fragments are so small that they will not be replicated in PCR.
Now that we added the DNase and it has done its job, how do we get rid of it, as it will also cleave the DNA we want to clone for PCR? There is also a very easy solution to this too! Just put it in an 80°C water bath, and watch it denature! This part is actually kinda cool to watch because the pfu solution goes from a relatively clear color to a very cloudy mixture as the DNase and other contaminating proteins denature (I wish I had taken some pictures).
After one more centrifugation to remove the precipitated proteins and such, we are now ready for the final step before the pfu can really be used, dialysis. Dialysis is the process of removing things from a solution, think blood dialysis. For our dialysis we used a centrifuge and put the pfu solution in a special tube with a filter in it that lets all molecules smaller than 10kDa slip through. Pfu is 95kDa so we do not need to worry about losing it. Not only does this dialysis remove some unwanted molecules, it also can be used to change the buffer that contains the pfu, since it is not in the most stable buffer for storage. When we centrifuge the solution, we want to make sure that there is at least some of the original solution left, otherwise we will blowout all the pfu through the filter membrane. Then, after the centrifugation, we add the storage buffer, centrifuge, add storage buffer, centrifuge, and keep doing that until we think the original buffer has been diluted enough. Next, we add glycerol, which helps to further stabilize the solution so we can freeze it, to the solution until it makes up 50% of it, and finally we are done! Not quite though, we still need to test if it actually works. Stay tuned next, next week, as I will be on spring break, to see if the pfu actually works!
Now that we added the DNase and it has done its job, how do we get rid of it, as it will also cleave the DNA we want to clone for PCR? There is also a very easy solution to this too! Just put it in an 80°C water bath, and watch it denature! This part is actually kinda cool to watch because the pfu solution goes from a relatively clear color to a very cloudy mixture as the DNase and other contaminating proteins denature (I wish I had taken some pictures).
After one more centrifugation to remove the precipitated proteins and such, we are now ready for the final step before the pfu can really be used, dialysis. Dialysis is the process of removing things from a solution, think blood dialysis. For our dialysis we used a centrifuge and put the pfu solution in a special tube with a filter in it that lets all molecules smaller than 10kDa slip through. Pfu is 95kDa so we do not need to worry about losing it. Not only does this dialysis remove some unwanted molecules, it also can be used to change the buffer that contains the pfu, since it is not in the most stable buffer for storage. When we centrifuge the solution, we want to make sure that there is at least some of the original solution left, otherwise we will blowout all the pfu through the filter membrane. Then, after the centrifugation, we add the storage buffer, centrifuge, add storage buffer, centrifuge, and keep doing that until we think the original buffer has been diluted enough. Next, we add glycerol, which helps to further stabilize the solution so we can freeze it, to the solution until it makes up 50% of it, and finally we are done! Not quite though, we still need to test if it actually works. Stay tuned next, next week, as I will be on spring break, to see if the pfu actually works!
Saturday, March 14, 2015
Increasing the Scale
Welcome back everyone! This week taught me about just how much waiting there can be for experiments. We decided that it was time to start increasing the amount of bacteria we were producing by ten fold, going from 100mL to 1L of bacteria. Much of my time was spent waiting for the bacteria to grow (about 2 hours after having grown it overnight), then waiting for the bacteria to induce (4 hours), and a bunch more waiting from processes like centrifugation or running a gel or waiting for something to heat/cool. There is just so much waiting! Although, all of this waiting did allow me to finally straighten up my notes and label my SDS-PAGEs on PowerPoint, which I had to learn how to use.
From some prior research that I had received from my on-site mentor Dr. Gustin, I found that the size of pfu is about 95kDa. But, the research that I had obtained that number from also used a different type of pfu; a type that has a histidine tag. Histidine is an amino acid that has an attraction to metals like Ni, nickel. This means that if a protein has a bunch of histidine attached to its end, it will stick to that metal, and thus purify itself because it will be the only protein sticking to the metal. The pfu that I am making does not have a tag, so we are planning on purifying it through simple heat treatment, aka making it so hot that all other proteins fall apart leaving pfu by itself as it is thermostable. As of right now, I have made a protein soup by sonicating the 1L culture of bacteria after centrifuging it down into a pellet and re-suspending it. We could use the pfu in PCR right now, but it is probably way too concentrated plus we do not know the proper dilutions, and it still has DNA from the E. Coli in it. Next week, I will remove the DNA by treating it with an enzyme called DNase and hopefully further purify my pfu. Thank you!
 |
| Was finally able to label this and make it look all pretty! All Sizes are given in kDa. |
From some prior research that I had received from my on-site mentor Dr. Gustin, I found that the size of pfu is about 95kDa. But, the research that I had obtained that number from also used a different type of pfu; a type that has a histidine tag. Histidine is an amino acid that has an attraction to metals like Ni, nickel. This means that if a protein has a bunch of histidine attached to its end, it will stick to that metal, and thus purify itself because it will be the only protein sticking to the metal. The pfu that I am making does not have a tag, so we are planning on purifying it through simple heat treatment, aka making it so hot that all other proteins fall apart leaving pfu by itself as it is thermostable. As of right now, I have made a protein soup by sonicating the 1L culture of bacteria after centrifuging it down into a pellet and re-suspending it. We could use the pfu in PCR right now, but it is probably way too concentrated plus we do not know the proper dilutions, and it still has DNA from the E. Coli in it. Next week, I will remove the DNA by treating it with an enzyme called DNase and hopefully further purify my pfu. Thank you!
Subscribe to:
Posts (Atom)