TITLE: DNA Extraction from Sterivex Filters
AUTHORS: Christopher Neil Thornton, William Brazelton

[DESCRIPTION]
Modified 2015 by the Brazelton Lab from protocols by Rika Anderson, Colleen Kellogg, Julie Huber, and Byron Crump. Incorporated some recommendations from Lever et al. (2015) Frontiers in Microbiology doi: 10.3389/fmicb.2015.00476.

[STEPS]
SECTION: Hot Lysis
2. Add 1.4 mL of DEB to each Sterivex with syringe and needle. Position the needle just below the mouth of the Sterivex so that it does not come back out the top. Do not fill to the top – stop when solution covers white filter. Possible Stopping Point. Store at ­20ºC
SECTION: Hot Lysis
2.1. Possible Stopping Point. Store at ­20ºC
SECTION: Hot Lysis
3. Place sterivex filter in 50mL tube with holes.
SECTION: Hot Lysis
4. Incubate at 65ºC for 30 mins. on Genemate spinning machine.
SECTION: Hot Lysis
5. Vortex each sterivex again (inside the Falcon tube) for 30 seconds. Bead Beating:
SECTION: Bead Beating
6. Using a syringe, withdraw fluid from each Sterivex and eject into bead tube (glass 0.1 mm for bacteria).
SECTION: Bead Beating
7. Bead beat for 40 s.
8. Centrifuge for 2 min at 5000 g.
9. Transfer fluid ­ avoiding beads ­ into fresh Eppendorf tube. Add no more than 900 μL in each tube (or no more than 750 μL if using 1.5 mL tubes). 
11. Gently shake a few times and then centrifuge at 14,000g for 1 minute.
12. Remove supernatant to fresh tube.
13. Add equal volume of chloroform / isoamyl alcohol (24:1) to each tube.
14. Gently shake a few times and centrifuge.
15. Remove supernatant to fresh tube, carefully avoiding the bottom organic layer. Ethanol precipitation:
SECTION: Ethanol precipitation
16. Redistribute aqueous phase among 3 tubes so that each 2.0 mL tube has 550 μL or less and each 1.6 mL tube has 450 μL or less. For some samples, additional salt is not necessary, and you can skip the sodium acetate. In this case, you can add up to 600 μL in a 2.0 mL tube.
SECTION: Ethanol precipitation
17. Add 0.1 volumes sodium acetate (3M, pH 5.2). (e.g. add 55 μL to 550 μL.)
SECTION: Ethanol precipitation
18. Add 2 volumes 100% ethanol. (e.g. add 1210 μL to 605 μL.)
SECTION: Ethanol precipitation
19. [optional for low biomass samples] Add 1.2 ul of glycogen (20 ug/ul).
SECTION: Ethanol precipitation
20. Invert a few times to mix.
SECTION: Ethanol precipitation
21. Incubate at ­20ºC for at least 1 hr. or overnight. Incubation on ice might work just as well and yield a cleaner pellet.
SECTION: Ethanol precipitation
22. Centrifuge for 40 minutes at 16,000g. (Optional: used cooled centrifuge at 0ºC)
SECTION: Ethanol precipitation
23. Pour out supernatant. Do not completely invert tube; keep at a gentle angle to minimize the chance of the pellet falling out.
SECTION: Ethanol precipitation
24. Add 500 μL of cold 70% ethanol to each tube.
SECTION: Ethanol precipitation
25. Invert the tube to mix. Make sure the pellet is dislodged from the bottom so that it is properly washed.
SECTION: Ethanol precipitation
26. Centrifuge at 16,000g for 10 minutes.
SECTION: Ethanol precipitation
27. Remove liquid again with pipettor. Be careful to avoid pellet.
SECTION: Ethanol precipitation
28. Place tubes with open lids in the Vacufuge. Spin for 7 minutes at 30ºC on the V­AL setting. If you can see ethanol in the tube, spin for another 2­5 minutes. If the pellets become powdery, they are too dry.
SECTION: Ethanol precipitation
29. Resuspend in ~100 μL of low EDTA TE. Heat to 55ºC 
 
for 10 or more minutes to dissolve pellet and store at 4ºC. For long­term storage, place at ­20 or ­80ºC, but avoid repeated freezing and thawing of the DNA. One strategy is to keep half at 4ºC for the working sample and store the other half at ­80ºC as the archive sample.
SECTION: Prepare DEB
1. Prepare DNA Extraction Buffer (DEB):
        0.1M Tris-HCl (pH 8)         4.5 mL of 1.0 M
        0.1M Na-EDTA (pH 8)          9 mL of 0.5M
        0.1M KH2PO4  (pH 8)     0.54 g
        1.5M NaCl                        13.5 mL of 5M 
0.8M Guanidine HCl                3.44 g
0.5% Triton-X 100                  0.225 mL (225 μL) of 100%

Add above ingredients to 50 mL tube. 
Add milli-Q water to ~40 mL
Add NaOH to pH 10 (several drops at a time)
Add milli-Q water to 45 mL
Filter-sterilize to remove possible spores
Autoclave. Slightly loosen lid so that it is not air-tight. Recover from autoclave very soon after the autoclave cycle is completed.
Pour autoclaved solution into fresh 50 mL tube.
Aliquot into 1.5 mL tubes.
SECTION: Phenol/Choroform Extraction
10. Add equal volume of phenol / chloroform / isoamyl alcohol (25:24:1, bought pre­mixed with alkaline buffer) to each tube.