Primers Primers everywhere and not a drop to drink

Today, (being this morning, afternoon, and fairly well into late tonight), has been spent diluting primers, and preforming PCR to see what I can see. Side note: just looked outside, and there is one hell of a thunderstorm raging on. It was pouring earlier, but abated long enough to make a quick run to home depot to get dinner with the boy (an easy 4 mile bike ride each way).

In any case, if PCR (Polymerase chain reaction: the way we copy DNA to do stuff with), was a zipper, primers would be the little crimped pieces of metal on the ends that tell the zipper where to start zipping, and where to stop zipping: they set the frame where the zipper can work. Also, to continue this analogy, PCR is like taking a zipper (a double helix of DNA), and heating it up so hot that the zipping mechanism and previously mentioned crimped metal ends falls off. This leaves two zipper strands. In PCR, the first step is to heat the DNA up so that it denatures (comes apart leaving 2 separate strands)...see the parallels?

Next, what we do is add primers to the DNA (or crimped metal ends to the zipper). This tells the DNA were we want it to replicate. These primers are pieces of DNA that are custom made so that they match up to certain segments of DNA that are present in all bacteria, or just some bacteria, which lets us be specific with what DNA we amplify. The other 2 important chemicals present in a PCR reaction, are the free nucleotides, the building blocks of DNA (think free individual zipper teeth), and Taq Polymerase, which is an enzyme that allows DNA to copy itself (for the zipper analogy, this equates to the zipper pull which physically connects the two sides of the zipper). The PCR reaction occurs at several different temperatures, which are cycled over and over again (more than 40 times) to grow DNA.

What happens is this:
First, the DNA is heated, this causes it to denature and separate into two strands (as mentioned earlier with the zipper)
Next, the temperature drops so that primers can attach to the DNA (think of putting those metal crimps on half of a zipper). This is called annealing.
After that, the temperature is raised again, and as this happens, DNA polymerase starts at the primers, and works along the DNA in one direction, grabbing free nucleotides from the liquid mixture, to match up to the DNA bases on the other side (imagine putting the zipper pull on the crimped metal end, and as you pull up to zip the half zipper, pieces of zipper are inserted into the other side of the pull to combine and make a complete zipper).

This cycle is repeated over and over again, each time doubling the DNA present in the reaction tube. Once this is done, then we run it on an agar gel (like jello), by applying an electric current that pulls the DNA along the charge (since DNA is negatively charged). This allows us to separate pieces of DNA that we amplified based on size.

Anyway, thats what I'm running up in lab now. I am in the process of getting a new anaerobic station. Here is a picture of the one that everyone in lab uses

The tubes on the left are used to run gasses into syringes to make solutions anoxic. Gasses are passed through a reduced copper column to remove all traces of oxygen, then aseptically added to syringes or solutions so that anaerobic bacteria can live!

My station is to the right: I picked out the awesome green contact paper to give it more of flair. i wanted to spray paint everything purple, but I decided that contact paper was the better option (err...I was told it was the better option)



Its still pouring rain, and I have another two hours until my PCR is done running. I'll come in and do the gel tomorrow. Hopefully there won't be anymore lightening as I still have to ride my bike home tonight.