PCR power

Yes, so I am still working on my project, which will involve 27 primer pairs and 12 sea urchin individuals. If you do the math, that's 324 different base pair sequences. Here's the process: PCR, gel electrophoresis, DNA purification, specs, and sequencing. For 324 different tubes. Needless to say, it will take some time, but I am almost finished PCRing all the tubes. Yesterday, I did an 8 by 12 plate of PCR reaction. Once we could get it to work, I used an automatic pipette to alloquate the primer master mixes into the 96 tubes. Surprisingly, PCR involves more math than I thought. It wasn't difficult, but I was actually forced to use a calculator. WHen I am creating the PCR master mixes, sometimes I feel like I'm cooking, putting together a recipe.. 4 ul of sugar, 25 ul of water, and 750 ul of baking soda. Delicious. But like cooking, you have to be careful about not putting in the correct amount of each ingredient. PCR is a reaction that involves a lot of different components, and depending on how many tubes you have, you master mix will be correspondingly bigger or smaller. The lab even has its own blender- the tabletop vortex that mixes up the ingredients to make everything homogenous. Of course, all this "biological cooking" hasn't changed my mind about kitchen cooking... I still avoid it at all costs, but I am learning that scientists have to be alert and focused to get specific reactions like PCR right. If you're not careful, you could spoil the batch and waste ingredients while you're at it. Overall, it's been an interesting week.

Lots of PCRs

This first week has gone by so fast. The undergrads in my lab tell me that 7 weeks will go by like 2 days. Now I have only 5 weeks left to do my project. Although I have a vague idea about what my project will be, I don't really have a hypothesis or specific question I am answering. I am simply investigating a bunch of different genes, PCRing them, sequencing them, and then noticing any point mutations, or SNPs, that fall out of it. Since sea urchins are have a high crossing-over frequencing, and therefore have a good deal of mutations. For my project, I will be looking at 12 individuals and 27 primer sequences that will create 27 different bp sequences anywhere from 100 to 1500 bp in length. These sequences are all coding sequences in the skeletogenic network of Strongylocentrotus purpuratus, a gene network that has been heavily researched and controls the development of the sea urchin's skeleton. I am looking at each of the 27 sequences in each individual, if I have time, in order to find any interesting mutation or mutations that this particular population has in common. Since each individual has 2 copies of its genetic material, I am actually sequencing 24 x 27 sequences of DNA, and that means 12 x 27 different PCRs. To make things quicker and easier, I am doing 4 individuals with 4 primers at a time, so that I do 16 PCRs at a time.
I also learned to use the spec machine on Friday. This allows you to look at how efficient the DNA purification after PCR was. It measures the concentration of DNA vs. protein, as well as the content of DNA vs. other junk that you don't want. You pipette a tiny droplet of the mixture onto a small metal surface and the machine uses light rays to read the DNA concentration in the water.
Overall, I had a very exciting week. I have learned so much, and gotten so much experience. It was a rush of different protocols and vocabulary, and I can't wait to learn more next week and the rest of this summer!