TITLE: RNA Extraction from Sterivex Filters
AUTHORS: William  Brazelton, H Lizethe Pendleton

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Modified 2020 by H. Lizethe Pendleton from the Brazelton Lab DNA extraction protocol.</div></div>

[STEPS]
?. [Prepare DEB]
Nucleic Acid Extraction Buffer (NEB):0.1M Tris-HCl (pH 8)            4.5 mL of 1.0 M0.1M Na-EDTA (pH 8)             9 mL of 0.5M0.1M KH2PO4  (pH 8)       0.54 g1.5M NaCl                           13.5 mL of 5M 0.8M Guanidine HCl          3.44 g0.5% Triton-X 100               225 μL of 100%Add above ingredients to 50 mL tube. Add UltraPure water to ~40 mLAdd NaOH to pH ~7 (slowly)Add UltraPure water to 45 mLFilter sterilize with 0.22 μm filterAutoclave with lid loosened. Recover from autoclave soon after the cycle is complete.Pour autoclaved solution into fresh 50 mL tube.Aliquot into 1.5 mL tubes.UV sterilize for 10-30 minutes.
?. [Hot Lysis]
Add 1.4 mL of NEB 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 the solution covers the filter.
?. [Hot Lysis]
Possible Stopping Point. Store at ­20ºC
?. [Hot Lysis]
Incubate at room temperature for 30 minutes.
?. [Hot Lysis]
Vortex each sterivex (inside a Falcon tube) for 30 seconds.
?. [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]
Transfer fluid - avoiding beads - into fresh Eppendorf tube. Add no more than 350 μL in each 1.7 mL tube.
?. [RNA Acidic Phenol/Chloroform Extraction]
To obtain RNA from the lysates:Add equal volume of acid phenol / chloroform (pH 5.2) to each tube.
?. [RNA Acidic Phenol/Chloroform Extraction]
Gently shake a few times and then centrifuge at 14,000g for 1 minute.
?. [RNA Acidic Phenol/Chloroform Extraction]
Remove supernatant (300 uL) to fresh tube. Avoid organic phase and interface. Repeat phenol extraction until the interface is clear. Keep the tube with the organic phase-- this is where the DNA (theoretically) should be.
?. [RNA Acidic Phenol/Chloroform Extraction]
Add equal volume of chloroform / isoamyl alcohol (24:1) to each tube.
?. [RNA Acidic Phenol/Chloroform Extraction]
Gently shake a few times and centrifuge.
?. [RNA Acidic Phenol/Chloroform Extraction]
Remove supernatant to fresh tube, carefully avoiding the bottom organic layer.
?. [Ethanol precipitation]
Redistribute aqueous phase among tubes:RNA: 250 μL in each tubeDNA: 400 μL in each tube
?. [Ethanol precipitation]
Add salt solutions:RNA: 0.5x volumes of ammonium acetate (7.5 M) (ex: 125 μL to 250 μL)DNA: 0.1 volumes of sodium acetate (3M, pH 5.2). (ex: 40 μL to 400 μL)
?. [Ethanol precipitation]
Add ethanol:RNA: 2.5x volumes of 100% ethanol (ex: 937.5 μL to 375 μL)DNA: 2.0 volumes of 100% ethanol  (ex: add 880 μL to 440 μL.)
?. [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 UV hood. Let sit until ethanol has evaporated, but pellets are still damp. If the pellets become powdery, they are too dry.
?. [Ethanol precipitation]
Resuspend nucleic acid pellets:RNA: 50-100 μL of UltraPure water, pipette to mix. If pellets don’t readily dissolve, incubate at 55ºC for 30-60 seconds, but avoid heating for too long as RNA may degrade.DNA: 50-100 μL of low EDTA TE. Heat to 55ºC for 10 or more minutes to dissolve pellet.
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]
This is a total nucleic acid extraction protocol that provides the option of obtaining both RNA and DNA in separate aliquots, though it is optimized for RNA. We have a separate protocol optimized for DNA.
?. [DNA Alkaline Phenol/Chloroform Extraction]
To obtain DNA from the same lysates:Add 300 μL UltraPure Water to the original tubes, shake gently to mix.
?. [DNA Alkaline Phenol/Chloroform Extraction]
Add equal volume (of total volume) of phenol / chloroform (pH 8.0) to each original tube.
?. [DNA Alkaline Phenol/Chloroform Extraction]
Gently shake a few times and then centrifuge at 14,000g for 1 minute.
?. [DNA Alkaline Phenol/Chloroform Extraction]
Remove supernatant to fresh tube.
?. [DNA Alkaline Phenol/Chloroform Extraction]
Add equal volume of chloroform / isoamyl alcohol (24:1) to each tube.
?. [DNA Alkaline Phenol/Chloroform Extraction]
Gently shake a few times and centrifuge.
?. [DNA Alkaline Phenol/Chloroform Extraction]
Remove supernatant to fresh tube, carefully avoiding the bottom organic layer.
?. [Ethanol precipitation]
Convert RNA to cDNA immediately and store remaining RNA in -80ºC freezer. Once converted to cDNA, a working aliquot can be stored at 4ºC for short term purposes, or -20ºC or -80ºC for long term storage. Avoid repeated freezing and thawing of RNA and cDNA.If RNA does not need to be converted to cDNA in the lab and is being prepped to be sent for RNA sequencing, quantify RNA and DNA present in RNA extract, and immediately continue on to concentrating/cleaning bead step. Avoid freeze/thaw cycles as RNA is highly unstable and will easily degrade.DNA working aliquots can be stored at 4ºC. For long-term 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.