Banana test
"We shall conquer the larvae. They may be small but shall not escape!"
- an email from Hanny
Alright, friends, so I got all the DNA that Hanny and I collected from young corals in Palau sent off for sequencing. However, there are still a few samples left. In addition to tissue chips we collected from coral colonies (the DNA I just sent off), we had collected a few coral larvae and young settlers called spat.
We don't have enough larvae and spat for a population-level genetic analysis, but we still want to get as much information as we can from the samples. By extracting the DNA from our larvae and spat, Hanny and I can identify them and see what species are dispersing and settling at each of our study sites. But that's just the thing - extracting DNA from larvae and spat is hard. There's so little tissue in a single individual that conventional methods don't work. I tried three different methods with no success, and I was wasting samples along the way. Time for a different strategy.
Time...for a banana test.
Fresh fruit has some of the highest DNA concentrations of any biological tissue. In fact, extracting DNA from strawberries and bananas is a great science lab to do with children. I needed an idiot-proof way to develop my own DNA extraction method, so I brought my breakfast banana into the lab and scraped off tiny flecks of the fruit with a sterile pipet tip.
I tried one standard DNA extraction kit and three variations on a less-established method. After searching in the scientific literature, I decided to throw in my own method as well, by combining reagents from two different kits. I added all the reagents to the tubes, went through the proper incubation steps, and then tested the results using a NanoDrop. The machine showed ambiguous results - could be DNA, could be protein, could be DNA with high concentrations of protein contamination. Interestingly, my pieced-together method showed the most promising result.
I decided to try with some larvae. Another WHOI scientist was kind enough to give me some anemone larvae for the test - they're similar to coral larvae and served as a good analogue. I tried my pieced-together method, plus the two other variations, but the NanoDrop again showed ambiguous results. I needed another way to test for the presence of DNA.
Here's where the story takes an ironic twist: the best way to test for DNA in my samples was to try and make copies of it using PCR - a procedure that until just a few weeks ago was driving me nuts. I've gotten the hang of it now, thanks to Hanny, and been able to use it to answer important questions. In this case, PCR was my friend.
I got a tried-and-true PCR recipe to use on the anemone DNA from Hanny. I combined the reagents and subjected them to the prescribed thermal cycles. I ran the samples out on an electrophoresis gel and used UV light to visualize the DNA.
Drumroll please....
It worked! The gel you see to the right here shows bright bands of DNA that was amplified during the PCR procedure. This could only have happened if there was DNA in the sample originally - meaning that my extraction worked!
I was very proud of myself for working out the DNA extraction procedure. I can't wait to use it on the coral larvae and spat!
- an email from Hanny
Alright, friends, so I got all the DNA that Hanny and I collected from young corals in Palau sent off for sequencing. However, there are still a few samples left. In addition to tissue chips we collected from coral colonies (the DNA I just sent off), we had collected a few coral larvae and young settlers called spat.
 |
| A coral spat on a terra cotta panel. The scale bar is in the bottom left, and as you can see, this settler is < 1 mm across. |
Time...for a banana test.
Fresh fruit has some of the highest DNA concentrations of any biological tissue. In fact, extracting DNA from strawberries and bananas is a great science lab to do with children. I needed an idiot-proof way to develop my own DNA extraction method, so I brought my breakfast banana into the lab and scraped off tiny flecks of the fruit with a sterile pipet tip.
I tried one standard DNA extraction kit and three variations on a less-established method. After searching in the scientific literature, I decided to throw in my own method as well, by combining reagents from two different kits. I added all the reagents to the tubes, went through the proper incubation steps, and then tested the results using a NanoDrop. The machine showed ambiguous results - could be DNA, could be protein, could be DNA with high concentrations of protein contamination. Interestingly, my pieced-together method showed the most promising result.
I decided to try with some larvae. Another WHOI scientist was kind enough to give me some anemone larvae for the test - they're similar to coral larvae and served as a good analogue. I tried my pieced-together method, plus the two other variations, but the NanoDrop again showed ambiguous results. I needed another way to test for the presence of DNA.
Here's where the story takes an ironic twist: the best way to test for DNA in my samples was to try and make copies of it using PCR - a procedure that until just a few weeks ago was driving me nuts. I've gotten the hang of it now, thanks to Hanny, and been able to use it to answer important questions. In this case, PCR was my friend.
 |
| My electrophoresis gel |
Drumroll please....
It worked! The gel you see to the right here shows bright bands of DNA that was amplified during the PCR procedure. This could only have happened if there was DNA in the sample originally - meaning that my extraction worked!
I was very proud of myself for working out the DNA extraction procedure. I can't wait to use it on the coral larvae and spat!
Comments
Post a Comment