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

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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.</div></div>

[STEPS]
?. [Hot Lysis]
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
?. [Hot Lysis]
Possible Stopping Point. Store at ­20ºC
?. [Hot Lysis]
Place sterivex filter in 50mL tube with holes.
?. [Hot Lysis]
Incubate at 65ºC for 30 mins. on Genemate spinning machine.
?. [Hot Lysis]
Vortex each sterivex again (inside the Falcon tube) for 30 seconds. Bead Beating:
?. [Bead Beating]
Using a syringe, withdraw fluid from each Sterivex and eject into bead tube (glass 0.1 mm for bacteria).
?. [Bead Beating]
Bead beat for 40 s.
?. [Bead Beating]
Centrifuge for 2 min at 5000 g.
?. [Bead Beating]
Add equal volume of phenol / chloroform / isoamyl alcohol (25:24:1, bought pre­mixed with alkaline buffer) to each tube.
?. [Phenol/Chloroform extraction]
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). Phenol / chloroform extraction:
?. [Phenol/Chloroform extraction]
Gently shake a few times and then centrifuge at 14,000g for 1 minute.
?. [Phenol/Chloroform extraction]
Remove supernatant to fresh tube.
?. [Phenol/Chloroform extraction]
Add equal volume of chloroform / isoamyl alcohol (24:1) to each tube.
?. [Phenol/Chloroform extraction]
Gently shake a few times and centrifuge.
?. [Phenol/Chloroform extraction]
Remove supernatant to fresh tube, carefully avoiding the bottom organic layer. Ethanol precipitation:
?. [Ethanol precipitation]
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.
?. [Ethanol precipitation]
Add 0.1 volumes sodium acetate (3M, pH 5.2). (e.g. add 55 μL to 550 μL.)
?. [Ethanol precipitation]
Add 2 volumes 100% ethanol. (e.g. add 1210 μL to 605 μL.)
?. [Ethanol precipitation]
[optional for low biomass samples] Add 1.2 ul of glycogen (20 ug/ul).
?. [Ethanol precipitation]
Invert a few times to mix.
?. [Ethanol precipitation]
Incubate at ­20ºC for at least 1 hr. or overnight. Incubation on ice might work just as well and yield a cleaner pellet.
?. [Ethanol precipitation]
Centrifuge for 40 minutes at 16,000g. (Optional: used cooled centrifuge at 0ºC)
?. [Ethanol precipitation]
Pour out supernatant. Do not completely invert tube; keep at a gentle angle to minimize the chance of the pellet falling out.
?. [Ethanol precipitation]
Add 500 μL of cold 70% ethanol to each tube.
?. [Ethanol precipitation]
Invert the tube to mix. Make sure the pellet is dislodged from the bottom so that it is properly washed.
?. [Ethanol precipitation]
Centrifuge at 16,000g for 10 minutes.
?. [Ethanol precipitation]
Remove liquid again with pipettor. Be careful to avoid pellet.
?. [Ethanol precipitation]
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.
?. [Ethanol precipitation]
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.
Recipe for low EDTA TE: 10 mM Tris­HCl 0.1 mM EDTA For 50 ml: 500 μl 1 M Tris­HCl (pH 8.0) autoclaved 10 μl 0.5 M EDTA (pH 8.0) autoclaved → to 50 ml with milliQ H2O → filter sterilize with 0.22 μm syringe filter TE is good for DNA storage, but EDTA inhibits PCR. So this low EDTA TE buffer is a good compromise for storing DNA for later PCR amplification. You can also just use EB (10 mM Tris­HCl, pH 8 or 8.5).
?. [Prepare DEB]
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 g0.5% Triton-X 100                  0.225 mL (225 μL) of 100%Add above ingredients to 50 mL tube. Add milli-Q water to ~40 mLAdd NaOH to pH 10 (several drops at a time)Add milli-Q water to 45 mLFilter-sterilize to remove possible sporesAutoclave. 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.