TITLE: DNA extraction from colonial tunicates
AUTHORS: Marta   Wawrzyniak, Simon  Blanchoud

[DESCRIPTION]
This protocol has been successfully used with Botrylloides diegensis and was adapted to our needs based on the HotPhenol DNA extraction protocol.


[GUIDELINES]
Change gloves frequently, particularly as the protocol progresses from crude extracts to more purified materials. Use sterile tubes. Perform all steps on ice and use RNAse-free and DNase-free water unless otherwise stated.

[STEPS]
SECTION: Cell Lysis
2. Add 500 µL of Ph-Ch-IA solution and 350 µL of SDS-Lysis buffer.

SECTION: Cell Lysis
4. Mix by vortexing at maximum speed for 45 s .

SECTION: Cell Lysis
5. Cool for 1 minon ice.     
SECTION: Cell Lysis
6. Mix again by vortexing at maximum speed for 45 s.  
SECTION: DNA extraction
11. Centrifuge at Room temperature at maximum speed for 3 min .

SECTION: Cell Lysis
7. Heat the tubes at 70 °C  for 10 min, mix regularly by inversion. 
SECTION: Cell Lysis
8. Mix again by vortexing at maximum speed for 45 s.  
SECTION: Cell Lysis
9. Cool for 1 minon ice.   
SECTION: Cell Lysis
10. Mix again by vortexing at maximum speed for 45 s.  
SECTION: Cell Lysis
3. Heat the tube at 70 °C  for 2 min . 
SECTION: DNA extraction
12. Transfer 400 µL of the upper aqueous phase to a new tube.

SECTION: DNA extraction
13. Add 400 µL of Ph-Cl-IA solution. 

SECTION: DNA extraction
14. Shake the tube by inversion for 30 s .

SECTION: DNA extraction
15. Centrifuge at maximum speed for 3 min .
SECTION: DNA extraction
16. Transfer 300 µL of the upper aqueous phase to a new tube.
SECTION: DNA extraction
17. Add 300 µL of Ph-Cl-IA solution. 
SECTION: DNA extraction
18. Shake the tube by inversion for 30 s .
SECTION: DNA extraction
19. Centrifuge at maximum speed for 3 min .
SECTION: DNA extraction
20. Transfer 200 µL of the upper aqueous phase to a new tube.
SECTION: DNA extraction
21. Add 200 µL of Cl-IA solution. 
SECTION: DNA extraction
22. Shake the tube by inversion for 30 s .
SECTION: DNA extraction
23. Centrifuge at maximum speed for 3 min .
SECTION: DNA extraction
24. Transfer aqueous phase to a new tube.
SECTION: DNA precipitation
25. Add 2 volumes of 100 % volume Ethanol (typically 300-400 µL ). 

SECTION: DNA precipitation
26. Add 0.1 volume of 3 Molarity (M) sodium acetate (typically 15-20 µL ).

SECTION: DNA precipitation
27. Mix by inversion. 
SECTION: DNA precipitation
28. Incubate at -20 °C for 180 min .

SECTION: DNA precipitation
29. Centrifuge at maximum speed for 20 min at 4 °C .

SECTION: DNA precipitation
30. Discard the supernatant. 
SECTION: DNA precipitation
31. Add 450 µL of cold 80 % volume ethanol. 

SECTION: DNA precipitation
32. Centrifuge at maximum speed for 5 min at 4 Room temperature .
SECTION: DNA precipitation
33. Discard the supernatant. 
SECTION: DNA precipitation
34. Add 200 µL of cold 80 % volume ethanol. 
SECTION: DNA precipitation
35. Centrifuge at maximum speed for 5 min at 4 Room temperature .
SECTION: DNA precipitation
36. Discard the supernatant. 
SECTION: DNA precipitation
37. Resuspend the pellet in ultra pure water (typically 20-100 µL ).

SECTION: DNA precipitation
38. Measure the DNA concentration using the NanoDrop.
SECTION: DNA precipitation
39. Store at -20 °C for short storage or at -80 °C for long storage.

1. This protocol was developed to extract both RNA and DNA in parallel (See RNA extraction from colonial tunicates , steps 1-7) using the same samples. However it could be run directly on fresh samples (steps 1.1-1.4).  
1.1. Clean the slide from which you will take the colony of your interest. See Cleaning colonial ascidians.
1.2. Isolate a cleaned colony composed of approx. 20 zooids. 
1.3. Transfer to a tube and spin at maximum speed for 2 min .
1.4. Remove the excess water.
SECTION: Cell Lysis
3.1.  If working with fresh samples, add glass beads 0.1mm to the tube and shake on eppendorf thermal shaker at 900rpm. 
SECTION: Cell Lysis
7.1.  If working with fresh samples, shake the tubes on eppendorf thermal shaker at 900rpm.