Wait for it

"I am the one thing in life I can control...

I'm not falling behind or running late
I'm not standing still
I am lying in wait"

- "Wait for it" from the musical Hamilton by Lin-Manuel Miranda

There is a lot of waiting in science. I have to wait for proposals to be reviewed, collaborators to contribute, papers to be published, technology to be usable, samples to be processed, paperwork to be cleared, and the list goes on and on. It was actually a little strange for me to come back from Palau and launch right into lab analysis of the samples I had just collected. We need the data from those samples by next May, so I could not afford to wait. 

The analysis that we're planning to do with those coral samples is called 2bRAD. It's a form of restriction site-associated DNA sequencing, which is a complete mouthful. In very simple terms, we're planning to chop up the DNA randomly, sequence the fragments, and find all the little places that the DNA sequence varies from one individual to another. Those little variations will show us which population each individual belongs to. We'll then go through and pick individuals from two different populations to include in our experiment next spring. 

2bRAD is an incredibly powerful technique, and I'm honestly pretty glad we can get such high-resolution identifications out of our coral samples. Here's the catch (there's always a catch): the DNA that you use in a 2bRAD analysis has to be pristine. Intact, clean, no other proteins or cellular structures or weird chemicals hanging on. Pristine DNA. Exactly 100 ng of it. 

Getting a DNA sample clean enough to use in 2bRAD is a lot of work - it occupied my days for the last two weeks. In order to extract DNA from a sample, you start by bursting all the cells, then selectively removing everything in the cellular soup that's not DNA. I took our samples through a standard DNA extraction kit that includes several cleaning steps, then put them through another multi-step cleaning kit. You lose some DNA in each of the cleaning steps, so if you don't have much in your cellular soup to begin with, you can accidentally wash it all away. There were a few samples that had no DNA left in the soup by the end, so I had to start completely over. 

Once you have ultra-clean DNA solution in a tube, you have to measure the concentration. 2bRAD requires exactly 100 ng of DNA in exactly 4 μL of water - that's a concentration of 25 ng/μL. A small minority of the samples were on the money; some had to be diluted (no big deal); and some were infuriatingly close - around 20 ng/μL. I wasn't sure what to do with the ones that were close but not perfect. I didn't want to start over, so I contacted my collaborator at Boston University. Thankfully, her lab has a vacuum system that can selectively evaporate water and condense dilute samples into smaller volumes. If there's 100 ng of DNA in the solution, she can get it down to 4 μL. I was relieved. 

I finished my work with the samples at the end of last week, packed them in ice, and sent them to Boston. My collaborator will take the baton from here and run the 2bRAD analysis in her lab. I'm proud of the work that I've done so far. Now the waiting begins.