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!