TITLE: SPOT DNA Extraction from 142mm A/E 1µm Filters
AUTHORS: Yi-Chun Yeh, Erin B Fichot, Alma E Parada

[DESCRIPTION]
Reference:  Lie et al, 2013. Small-scale temporal and spatial variations in protistan community composition at the San Pedro Ocean Time-series station off the coast of southern California. Aquatic Microbial Ecology 70, 93-110.

[BEFORE_START]
Preparation (prepare all solutions with DI water, then autoclave before use): 
1. 0.25 M EDTA, pH 8
2. 1 M Tris (Trizma base), pH 8
3. 2.5 M NaCl
4. 10% CTAB (hexadecyltrimethylammonium bromide)
5. 10.5 M ammonium acetate
Exception!
6. 10% SDS (w/v). Prepare with autoclaved DI water. DO NOT AUTOCLAVE! 

[STEPS]
SECTION: Day1
1. Prepare a 70°C water bath.


 


SECTION: Day1
2. Prepare a razor blade, forceps, and a metal slab.
 

SECTION: Day1
3. Cut the 142mm A/E filter into pieces on the metal slab and place it into a 50 ml falcon tube.

 

SECTION: Day1
4. Add 6.2 ml of 2X lysis buffer and 500 µl of 0.5 mm zirconia/silica beads.

 

 

SECTION: Day1
5. Bead-beat (vortex) for 1 min and then place in 70°C water bath for 7 min. Repeat this step 4 times.
SECTION: Day1
6. Spin at 3901 rcf in the swinging bucket rotor (#4250) on Beckman Allegra at 20°C for 2 min.
SECTION: Day1
7. Transfer the lysate to an Oak Ridge tube (labeled with “a”)
SECTION: Day1
8. Add 6.2 ml of 2X lysis buffer on the filter (still in the original 50 ml falcon tube) and repeat step 5 again to increase yield.
 

SECTION: Day1
9. Transfer the lysate to a new Oak Ridge tube (labeled with “b”) with a 20 ml syringe.

 

 

 

SECTION: Day1
10. Ideally, "a" and "b" Oak Ridge tubes both have 6.2 ml of lysate, but you may get more lysate from "b". In this case, pour "b" lysate to "a" to equalize the volume. 
SECTION: Day1
11. Add 1 ml of 10% CTAB (hexadecyltrimethylammonium bromide) and 2.8 ml of 2.5 M NaCl.

 


SECTION: Day1
12. Incubate in a 70°C water bath for 10 min.
SECTION: Day1
13. Add 28 ml of ice‐cold 100% EtOH (200 proof, molecular grade) and 3 ml of 10.5 M
ammonium acetate.
SECTION: Day1
14. Invert to mix thoroughly. 
SECTION: Day1
15. Store at ‐20°C overnight. 
SECTION: Day2
16. Spin at 24,000 g / 13,000 rpm in the swinging bucket rotor on Beckman Avanti JE or Beckman
Sorvall at 4°C for 2 hrs.

 

SECTION: Day2
17. Gently pour out supernatant and dry pellet upside‐down in the fume hood for at least 2 hours.

 

SECTION: Day2
18. Resuspend the pellet in 500 µl sterile 1X TE buffer for 2 hours at 37ºC. 
SECTION: Day2
19. Transfer to 2 ml non‐LoBind tubes.
SECTION: Day2
20. Add 500 µl phenol (bottom layer). Mix several times by gentle inversion.

 

 

 

SECTION: Day2
22. Remove the phenol (bottom layer) but leave the interface behind.
 

 

SECTION: Day2
23. Add 300 µl phenol and 300 µl chloroform:Isoamyl alcohol to the aqueous phase.  
SECTION: Day2
21. Spin for 2 min at 12000 rpm (13,000xg). 
SECTION: Day2
24. Mix several times by gentle inversion. Then spin for 2 min at 12000 rpm (13,000xg). 

SECTION: Day2
25. Remove the bottom layer but leave the interface behind.

 

SECTION: Day2
26. Add 500 µl chloroform:Isoamyl alcohol.
SECTION: Day2
27. Mix by gentle inversion. Then spin for 2 min at 12000 rpm (13,000xg). 
SECTION: Day2
28. Transfer the TOP layer (YOUR DNA) to a new 2ml non‐LoBind tube. LEAVE THE INTERFACE BEHIND. This top layer is your DNA!!!
SECTION: Day2
29. Add 1375 µl ice‐cold 100% EtOH (200 proof, molecular grade) and 125 µl 10.5M ammonium acetate. 
SECTION: Day2
30. Mix by inversion 5‐10 times. 
SECTION: Day2
31. Store at ‐20°C overnight. 
SECTION: Day3
32. Spin at 20,000 g on Beckman Avanti JE at 4°C for 2 hrs.
SECTION: Day3
33. Decant and air-dry the pellet in a bio-safety hood for 2 hrs.

 

SECTION: Day3
34. Resuspend the pellet in 30-50 µl sterile 1XTE buffer.
 

SECTION: Day3
35. Vortex and quick spin twice. Then, incubate at 40 °C for 1 hr.
SECTION: Day3
36. After re‐suspension, immediately quantify using PICO Green (Invitrogen).  
SECTION: Day3
37. If "a" and "b" both get good yields, pool "a" and "b". Then transfer half of the total volume to a 1.5 ml LoBind tube for archiving, and transfer another half to a 1.5 ml LoBind tube for working stock.