Porites astreoides DNA extraction continued

Continuing the extraction from yesterday, 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). DNA was resuspended in 0.2 ml nanopure water.

Here is the electrophoresis protocol I used:

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 an 8 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 5 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.


There were no bands present for either sample. Perhaps the DNA was degraded; the samples were collected 5 months ago. I did notice that both DNA pellets were quite brown in color.


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