I extracted DNA from the Anthopleura elegantissima heat stress experiment samples using DNAzol. This was done on 2/24/15 for samples from experiment 1, and on 3/3/15 for samples from experiment 2. See previous posts for details of these experiments.
The DNAzol protocol was as follows:
1. Tentacles were homogenized with a sterile plastic pestle in a microfuge tube with 0.5 ml DNAzol. I tried to apply as few strokes as possible, but anemone tentacles are very tough and are difficult to homogenize even after being frozen.
2. The homogenate was sedimented by centrifugation at 10,000 x g for 10 min. The supernatant was then transferred to a new tube.
3. DNA was precipitated by the addition of 0.25 ml 100% ethanol. Samples were mixed by inversion. A cloudy precipitate was generally observed, but few samples had noticeable threads of DNA formed. Thus, spooling was not attempted.
4. Samples were centrifuged at 4,000 x g for 1.5 min to pellet DNA. Supernatant was then aspirated.
5. DNA was washed twice with 1 ml 75% ethanol. A quick spin was used each time to pellet the DNA before removing the supernatant.
6. Samples were stored under refrigeration in 95% ethanol.
7. On 4/3/15, samples were centrifuged at 4,000 x g for 1.5 min to pellet DNA. The ethanol was aspirated and the samples were air dried under the hood for about an hour (they were watched to ensure they were not over dried).
8. DNA was resuspended in 0.2 ml nanopure water.
Yesterday (4/3/15), I finally got around to running a gel to take a look at the DNA. Many thanks to Jake for showing me where things were and getting me started. Here is the electrophoresis protocol I followed:
1. 75 ml TAE with 0.65 g agarose was microwaved for 1 min (with cap resting on 100 ml pyrex jar, watching for boil over) to heat and facilitate dissolution of the agarose. The jar was swirled with cap on. Solution was re-heated for a few more seconds to further facilitate particulate dissolution.
2. Solution left to cool without lid for 1-2 min
3. 7.5 ul EtBr added and swirled to mix
4. Solution left to cool for another minute before slowly pouring into the gel tray. The gel tray included a 12 slot comb, and the gel solution was poured in the opposite end from the comb.
5. Gel was left for 30 min to cool and harden.
6. Comb was removed and gel tray was rotated 90 degrees to align with electrodes.
7. Gel chamber filled with enough 1x TAE to just cover gel (~1 mm depth over gel).
8. Gel loaded with 20 ul green ladder (GeneRuler 100 bp) on each end. 20 ul of each sample was mixed with 3 ul purple loading dye on a sheet of parafilm and loaded into gel.
9. Electrodes plugged in and power supply set to 120 V.
10. Gel was run for 55 min, when ladder reference dye was ~1cm from edge of gel.
Sample abbreviations: L = ladder, A = ambient, H = heated, 5 = 5°C, 37 = 37°C
The very faint bands suggest very low concentrations of DNA; experiment 2 samples had darker bands, but they were still faint. Experiment 2 was performed in part because I suspected that not enough DNA was obtained from the 1-2 tentacles collected in experiment 1. So experiment 2 should have higher concentrations of DNA, as seen here, but apparently even more tissue would have been better. There is 0.18 ml sample remaining.
