Genomic DNA extraction Dinophilus 

Chema picked 250 adult Dinophilus on 02/03/14 and snap froze them on 03/10/14. We began the extraction following the S9 protocol. Buffers and solutions were already prepared by Chema on March. Steps are detailed below.

  1. Added 400 µl of GTC with 2-mercaptoethanol to Dinophilus tube.
  2. Animals were not dissolved 30 minutes after. Pistil did not help, so we left for 2 hours on ice.
  3. We split the volumes into 2 tubes to make it easier for balancing (200 µL each). We added 400 µL of ice-cold EtOH 100% to the tube and mixed gently by inverting the tubes.
  4. Left 30 minutes on ice.
  5. Spinned 20 minutes 13000 rpm @ 4°C.
  6. Removed supernatant of both tubes and washed the pellet with 1 ml EtOH 70%.
  7. Spinned 5 minutes 13000 rpm @ 4°C.
  8. Removed supernatant, spinned and collected the last drops and let them airdry.
  9. Resuspended the pellet with 300 µl of lysis solution with proteinase K.
  10. Incubated overnight @ 50°C.

09/10/2014

  1. Tissues were completely digested by the morning.
  2. We merged the two tubes into one (600 µL total) and extracted with one volume of Phenol/ChCl3 buffered @ pH 10 (600 µL).
  3. Tubes were sealed with parafilm and mixed for 30 min in the nuttator at max speed inside plastic container.
  4. Spinned 5 minutes 13000 rpm @ 25°C.
  5. I collected the upper layer into a new tube, but there was contamination from the organic layer. I then centrifuged again and re-collected the upper layer into another fresh tube. There was 20 µL left in the original collected tube which was centrifuged again and re-collect into the clean tube with the aqueous layer.
  6. Backed extracted the bottom layer with one volume of lysis solution without proteinase K and repeated steps 12, 13 and 14.
  7. Aqueous phase from all tubes was collected in a single tube, inverted once and split into 2 (500 µL each).
  8. Added 500 µL of chloroform and mixed for 30 min in the nuttator as before.
  9. Spinned 5 minutes 13000 rpm @ 25°C.
  10. Collected the upper layer into 2 separate tubes. One had 400 µL and the other 300 µL.
  11. Added 1/10 volume of Sodium Acetate 3M pH 5.5 (40 and 30 µL) and 2 volumes ice-cold 100% EtOH (800 and 600 µL), homogeneized them by inversion.
  12. Solution became a bit cloudier, but not much could be seen.
  13. Left at -20 freezer overnight at 1300.

10/10/2014

  1. Spinned 20 minutes 13000 rpm @ 4°C. There were pellets in both tubes.
  2. Removed supernatant from both tubes, washed with 1 ml EtOH 70%..
  3. Spinned 5 minutes 13000 rpm @ 4°C.
  4. Removed supernatant, spinned briefly to collect last drops and airdried.
  5. Added 10 µl of RNAse buffer to each tube, centrifuged briefly and waited 5 min so that the pellet completely dissolved.
  6. Merged tube contents together and measured the concentration in the NanoDrop. It was 236.8 ng µL (=4.7 µg in total).

Dgyr_DNA_beforeRNAse

  1. Added 180 µL of RNAse buffer and RNAse A to the 20 µL of DNA into a final concentration of 20 µg/ml. Tube was incubated for 1h15 @37°C.
  2. Extracted with one volume (200 µL) of Phenol/ChCl3 buffered @ pH 10.
  3. Tube was sealed with parafilm and mixed for 30 min in the nuttator at max speed inside plastic container.
  4. Spinned 5 minutes 13000 rpm @ 25°C.
  5. I collected the upper layer into a new tube.
  6. Backed extracted the bottom layer with one volume of lysis solution without proteinase K and repeated steps 12, 13 and 14.
  7. Collected upper layer again (300 µL).
  8. Added 300 µL of chloroform and mixed for 60 min in the nuttator as before.
  9. Spinned 5 minutes 13000 rpm @ 25°C.
  10. Collected the upper layer into 300 µL.
  11. Added 1/10 volume of Sodium Acetate 3M pH 5.5 (30 µL) and 2 volumes ice-cold 100% EtOH (600 µL), homogeneized them by inversion.

11/10/14

  1. Spinned 20 minutes 13000 rpm @ 4°C.
  2. Removed supernatant from both tubes, washed with 1 ml EtOH 70%.
  3. Spinned 5 minutes 13000 rpm @ 4°C.
  4. Removed supernatant, spinned briefly to collect last drops and airdried.
  5. Resuspended in 23 µL of the elution buffer of the gel extraction kit.
  6. Measured 1.5 in the NanoDrop 218 ng/µL (4.3 µg) and kept 1.5 µL for running a gel.

Dgyr_DNA_afterRNAse