Finale

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.

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 was to demonstrate the difference in the amount of DNA produced to the amount of pfu used

This PCR was also finally able to settle that adding BSA to the PCR solution does not change the amount of DNA produced.

There is barely any difference between these two.

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!

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.

Ooooooooh Science!

 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.

More Science!

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!

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.

It looks more pure than last time, the other stuff in it will not affect anything, probably.

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 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.

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!