Coral crusher: part 3
It is Saturday afternoon, and I am in my lab. My thighs and knees have that stiff-sore feeling you get after standing for hours, and my shoulder muscles are tense. I tilt my head side to side to stretch out, and I can feel a knot in the middle of my right shoulder blade. Out my corner window, I can see the hazy gray sky and the white masts of sailboats anchored across Eel Pond. Inside, you'd never know it was a weekend. I've been busy today.
I've been working on coral samples Hanny and I collected in Palau. First, we extracted DNA from all of the tissue chips we collected. Then, we measured the quality and concentration of the extracted DNA. As you might remember, we collected two different species of corals - a brain coral (Porites lobata) and a branching coral (Acropora sp.).
Ok, so Porites lobata is easy to identify. It's a big yellow sphere; it's everywhere; it's distinct; you can't miss it. But branching corals are much trickier. Hanny and I did our absolute best to only collect samples from one species while we were in Palau, but branching corals are really difficult to identify by eye. We might have grabbed two or three species without even knowing it. In order to see if our Acropora sp. samples belong to the same species, we have to use genetics.
So I set to work. I'm using a technique called the Polymerase Chain Reaction (PCR) to make copies of certain parts of the corals' DNA. PCR is a really powerful tool to zoom in on one region of the DNA because it makes millions of copies of the part you want while ignoring the rest. The region that's replicated is determined by what primers you use. Primers are short pieces of DNA that match to the region you want. During PCR, the two strands of DNA are pulled apart, and the primers attach to the beginning of the sequence you want to replicate. The primers act as guides to show the enzymes where to go so that only the target region is amplified.
Sounds easy enough, right? Wrong. The problem with PCR is that a hundred things can go wrong, and there are a hundred ways to fix each of them. Troubleshooting is a tangled nightmare. If a PCR fails, maybe your original DNA was bad. Maybe there was some salt in the solution that prevented the DNA from replicating. Maybe you added too much water. Maybe one of your reagents was degraded. Maybe you didn't keep the sample at the correct temperature or maybe you didn't leave it there for long enough or maybe this or maybe that or maybe the coral just doesn't like you.
I have run 4 different rounds of PCR with 5 different kinds of primers and 16 different samples from 3 projects including this one over the past 2 weeks. That's a total of 80 different little tubes of DNA, and guess how many of them worked? None.
I vented my frustration to Hanny, and she responded as always with a cool head and wisdom. "Molecular biology is a cruel game," she reassured me, "it's not you."
The good news is that at least for the corals, we have figured out the problem. We were using old primers borrowed from Hanny's postdoc advisor, and we believe the primers were degraded. They didn't leave their signature band on our electrophoresis gel, which means they weren't intact. Without primers to start the DNA replication process, the PCR didn't work.
We've ordered new primers and will give it another go as soon as they show up. With two steps forward and one step back, our project slowly progresses.
I've been working on coral samples Hanny and I collected in Palau. First, we extracted DNA from all of the tissue chips we collected. Then, we measured the quality and concentration of the extracted DNA. As you might remember, we collected two different species of corals - a brain coral (Porites lobata) and a branching coral (Acropora sp.).
Ok, so Porites lobata is easy to identify. It's a big yellow sphere; it's everywhere; it's distinct; you can't miss it. But branching corals are much trickier. Hanny and I did our absolute best to only collect samples from one species while we were in Palau, but branching corals are really difficult to identify by eye. We might have grabbed two or three species without even knowing it. In order to see if our Acropora sp. samples belong to the same species, we have to use genetics.
So I set to work. I'm using a technique called the Polymerase Chain Reaction (PCR) to make copies of certain parts of the corals' DNA. PCR is a really powerful tool to zoom in on one region of the DNA because it makes millions of copies of the part you want while ignoring the rest. The region that's replicated is determined by what primers you use. Primers are short pieces of DNA that match to the region you want. During PCR, the two strands of DNA are pulled apart, and the primers attach to the beginning of the sequence you want to replicate. The primers act as guides to show the enzymes where to go so that only the target region is amplified.
Sounds easy enough, right? Wrong. The problem with PCR is that a hundred things can go wrong, and there are a hundred ways to fix each of them. Troubleshooting is a tangled nightmare. If a PCR fails, maybe your original DNA was bad. Maybe there was some salt in the solution that prevented the DNA from replicating. Maybe you added too much water. Maybe one of your reagents was degraded. Maybe you didn't keep the sample at the correct temperature or maybe you didn't leave it there for long enough or maybe this or maybe that or maybe the coral just doesn't like you.
I have run 4 different rounds of PCR with 5 different kinds of primers and 16 different samples from 3 projects including this one over the past 2 weeks. That's a total of 80 different little tubes of DNA, and guess how many of them worked? None.
I vented my frustration to Hanny, and she responded as always with a cool head and wisdom. "Molecular biology is a cruel game," she reassured me, "it's not you."
The good news is that at least for the corals, we have figured out the problem. We were using old primers borrowed from Hanny's postdoc advisor, and we believe the primers were degraded. They didn't leave their signature band on our electrophoresis gel, which means they weren't intact. Without primers to start the DNA replication process, the PCR didn't work.
We've ordered new primers and will give it another go as soon as they show up. With two steps forward and one step back, our project slowly progresses.
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